[Frontiers In Bioscience, Landmark, 24, 133-171, Jan 1, 2019]

What does “NO-Synthase” stand for ?

Jerome Santolini1

1
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Universite Paris-Saclay,
F-91198, Gif-sur-Yvette cedex, France

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Distribution of NOS
3.1.Mammalian NOSs as exclusive NO-synthase models
3.2. Emergence of a new family of proteins
3.3. Prokaryotes, Eubacteria and Archae
3.4. Eukaryotes: fungi and plants
3.5. Metazoan
4. A new and heterogeneous family of proteines
4.1. The impasse of standard phylogenetic analysis
4.2. A singular versatile enzyme
4.2.1. NOS function
4.2.2. Instability of NOS activity and function
4.2.3. Overlaps of NOS activity
4.2.4. Multiplicity of NOS
4.2.5. What does NOS stand for?
4.3. The necessity of an original approach
5. Diversity of NOS structures
5.1. A variable assembly of multiple modules
5.2. Existence of other types of NOSs
5.3. Types of NOSs are not uniform within a simple phylogenetic group
5.4. Strong disparities in the structure of oxygenase domains
5.4.1. Basal metazoans
5.4.2. Plants
5.4.3. Cyanobacteria
6. Discussion: Diversity of functions
6.1. A Name is not a function
6.2. A Structure is not a function
6.2.1. A built-in versatile catalysis
6.2.2. A highly-sensitive chemical system
6.2.3. Electron transfer (ET) as a major NOS fingerprint
6.3. An Activity is not a function
6.3.1. The role of environment in NOS/NO activity
7. Conclusion: which function and which evolution for NOS?
8. References

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1. ABSTRACT
Mammalian NO-Synthases (NOSs) are
the enzymatic sources of Nitric Oxide (NO°), a
paradigmatic gasotransmitter involved in many (patho)-
physiological processes. The increasing number of
available genomes led to the identification of hundreds
of new NOS proteins throughout the kingdoms of
life, calling for a global investigation of this family of
proteins. These new NOSs are commonly believed
to share the same structure, functioning and role
as mammalian NOSs. The scope of this article is to
highlight the singularity of these NOSs and to describe
their complex structural and functional diversity. NOS
appears as a unique enzymatic machinery that exhibits
a complex Structure – Activity – Function relationship.
Its sophisticated redox mechanism and enzymatic
regulation, coupled to the vast biological chemistry of
reactive nitrogen species, leads to a specific cross-talk
between NOS catalysis and its biological environment
that implies a complex evolution of NOS function. This
paper addresses the relationship between structure,
function and evolution of NOS proteins using three
NOS model families and advocates for an integrative
and interdisciplinary approach that combines modelling
studies, structural characterization, and in vitro/in vivo
functional investigations.
2. INTRODUCTION
The biology of Nitric Oxide (NO°) started
almost 250 years ago with the chemical synthesis of
“Nitrous air” (NO°) by Joseph Priestley (1) and of its
dephlogisticated or diminished product (now known as
nitrous oxide, N2O). Although N2O biology was later on
investigated by Humphry Davy (2), NO° itself did not
gather much interest . 100 years later, another nitrogen
oxide emerged in human physiology: Amyl nitrite was
found to open the coronary arteries (Lauder Brunton,
(3)) and derivatives such as nitroglycerin were rapidly
used as medicine. It was even prescribed to Alfred
Nobel who was suffering from intense chest pain –
“Isn’t it the irony of fate that I have been prescribed
N/G 1, to be taken internally! They call it Trinitrin, so as
not to scare the chemist and the public” (letter dated
October 25, 1896 https://www.nobelprize.org/alfred_
nobel/biographical/articles/ringertz/). Another century
was necessary to unveil the major importance of NO°
and other reactive nitrogen species in Biology.
Although its biosynthesis was already
reported in bacteria as early as in the 50’s (4, 5), NO°
has long been considered as a poisonous chemical.
The concrete birth of NO° physiological history truly
coincides with the unveiling of the nature of the
Endothelium-Derived Relaxing Factor (EDRF (6,
7)). The discovery that such a major physiological
mediator could be a highly reactive, hydrophobic,
redox, diffusible gas somehow changed the way the
134
community envisions signaling processes (8-11). This
momentum has shaped the vision that we have of this
molecule and changed its biological status: from a
toxic, pollutant gas, it became a genuine biomolecule
that diffuses and reacts with its expected targets, a
unique mediator at the core of inter-cellular signaling.
Because of this historical mold, NO° field mostly
expands in mammals and reveals the involvement
of NO° in an always larger number of physiological
processes (12-15). Soon the enzymatic source of NO°
was identified, and this new family of proteins was
naturally named after their related production: NO-
Synthases. Three isoforms were characterized: NOS1
and NOS3 are constitutively expressed NOSs named
after the tissues they were isolated from: neuronal
NOS (nNOS, (16)) and endothelial NOS (eNOS,
(17)), respectively. NOS2 is inducibly expressed upon
immune signaling and was therefore named inducible
NOS (iNOS, (18)). These three types of NOSs were
almost exclusively investigated from proteins issued
from model mammals, respectively rattus norvegicus
(19), mus musculus (20) and bos taurus (21). These
three mammalian NOSs became the canonical
NOSs (22-26) on which most of the enzymological,
biochemical and structural investigations had been be
achieved (with some data on human NOS isoforms,
and splice variants (27-29)).
The standard knowledge acquired on NOSs
structure, activity and function, that we will now briefly
described, is entirely derived from investigations on
these sole mammalian NOSs. As this article does not
aim at providing a review on NOS structure, function
or activity, we invite readers to look for the most recent
reviews on that topics (30-36). Mammalian NOS
isoforms share a high sequence homology (37, 38).
All isoforms comprise an N-ter-oxygenase domain
(Oxy), a Calmodulin-binding region (CaM) and a C-ter
reductase domain (Figure 1), folded into a dynamic
dimeric structure (39-41). NO° synthesis is achieved
by the catalytic site, a heme-b cofactor buried within
the oxygenase domain (42) that catalyzes two
sequential oxidation reactions of L-arginine into
NO° and citrulline (Figure 2). Electrons required for
catalysis are conveyed by the reductase domain from
the second substrate (NADPH), through two flavin
cofactors (FMN and FAD), down to the heme catalytic
site (34, 43, 44). Calmodulin binding, upon increase
in Ca2+ concentration, allows a faster and coupled
electron transfer (ET) from one monomer’s FMN to the
other monomer’s oxygenase (Figure 1, (45-47)). Major
structural differences between mammalian NOSs are
both located in the N-ter and C-ter regions (24): i)
whereas iNOSs remains cytosolic upon transcription
and traduction, eNOS harbors palmitoylation and
myristoylation sites in the N-ter region, that anchors it
to the membrane of caveolae (48) and nNOS display
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Revisiting the structure, function and evolution of NOS family

Figure 1. Schematic representation of the NO-synthase quaternary structure. The reductase domain (ellipse) of one monomer (green) conveys the
electron provided by NADPH to the oxygenase domain (circle) of the other monomer (blue) via successive electron transfers through FAD, FMN. The
use of two sequential flavins allows the conversion of NADPH-hydride (H-) into regulated electron transfer up to the heme. This functional dimerization is
insured by the binding of Calmodulin (CaM, orange) to the CaM-binding region and by the binding of BH4 cofactor at the oxygenase interface.

Figure 2. The two consecutive catalytic steps of NO-synthase. L-Arginine hydroxylation requires one molecule of oxygen and one equivalent NADPH
leading to the formation of Nω-hydroxyl-arginine (NOHA). Mechanism is supposed to be similar to that of Cytochromes P450. NOHA oxidation follows a
NOS-specific mechanism that requires a second molecule of oxygen and a single external electron (0.5. NADPH).

as N-ter extension PDZ motifs that favors its binding
to NMDAR complexes (49, 50); ii) whereas iNOS ET
is mostly controlled by substrate binding (51), eNOS
and nNOS reductase domains display different Auto-
Inhibitory Elements (AIE) that finely tune and regulate
ET and heme reduction (33, 52); iii) another major
difference is the absence of sensitivity of CaM binding
towards Ca2+ for iNOS (53, 54). These differences
naturally fitted the distinction between highly regulated
constitutive NOSs devoted to signal transduction
– a process that requires fine catalytic tuning – and
unleashed iNOS, associated to cytotoxic activity, for
which a high and unregulated NO° production was
expected. Thus, the cleavage between iNOS and
constitutive NOSs functions has been mostly thought
of in terms of differences in NO° flux, protein/protein
interactions and post-translational modifications.
However, the study of NOSs molecular
mechanism makes this dual partition more complex as
it reveals sophisticated and specific catalytic regulation
patterns. This is mostly due to the particularity of the

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subtle redox mechanism of NOS oxygenase: although
NOS is fitted to produce NO°, NO° remains a strong
inhibitor of NOS (Ki in the pM range, (55, 56)) that is
engaged in fast geminate recombination (57). This
NO rebinding modifies NOS catalytic production by
generating two alternative catalytic cycles (31, 58,
59). In this regard, subtle changes in the values of
key-kinetic steps will differentially modify the catalytic
activity of NOS isoforms (55, 56). This is for example
the case for the ratio between NO° off-rate versus heme
reduction rates, the differences in ET rate between
isoforms, the variations in FeIINO oxidation rates (55,
58, 60-63). These small variations, unpredictable from
the 3D structures, induce differences in the NADPH/
NO coupling and in the sensitivity to external factors
(O2 concentration, Arg availability, redox status, (55,
58)). In the recent years, it has become obvious that
NOSs are a complex machinery that might catalyze
additional reactions (64-67) and produce many
different reactive oxygen species such as superoxide,
peroxynitrite…(60, 68-72).
Though, this knowledge has been overlooked.
Data have been produced faster than they had been
incorporated in an integrated picture of NOS/NO°
activity and function. The impressive improvement
of sequencing technologies has provided thousands
of new genomes in which the presence of proteins
homologous to NOS has been sought for. New
NOSs emerged at first from genomes from bacteria,
amoeba, mollusks, insects, and etc. X-Ray structures
of the oxygenase domains were generated almost
simultaneously (73, 74) or even before any functional
characterization (75). A few enzymatic (73, 76-78) or
mechanistic (79) assays confirmed the capacity of their
catalytic site to produce NO°, leading to the conclusion
that these many different NOSs were genuine NO-
Synthases. This shaped the belief and the practice of
the NOS/NO° community: any newly identified NOS
that harbors a similar oxygenase domain is believed to
behave like a mammalian NOS before any functional
characterization. This assumption is mobilized in most
of the investigations on NOSs. And even when these
NOSs do not seem able to produce NO°, I’ve been
replied, for example for bsNOS, that “they have to, it’s
just that we don’t know how they do it”…
This article aims at clarifying and improving
the way the NO° community deals with these many
new NO-Synthases and addresses the question about
“what NOS really stands for”. As we will try to show in
this article, the NOSs do not constitute a homogenous
protein family. This article provides a state-of-the-art
picture of the presence of NOSs in the whole tree of
life, highlighting their surprising structural diversity,
their large of array of – often opposite -- functions
and their inexplicable phylogenetic distribution. We
believe that the current knowledge and concepts in
the field are no longer adapted to investigate this new
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and vast scientific territory. In this regard, this article
will briefly describe NOSs from three distinct model
families: cyanobacteria, plants and basal metazoan
and discuss the words, concepts, paradigms we may
need to answer an apparently simple question: What
is a NOS ?
3. DISTRIBUTION OF NOSs
3.1. Mammalian NOSs as exclusive NO-synthase
models
The historical circumstances of the unveiling of
NO° physiological role – its implication in the regulation
of mammalian vascular tone - have delineated the field
in which NOS/NO° investigations were to be achieved
and circumscribed the research on NO-Synthases
mostly to the mammalian phylum. The first identified
NO-Synthases were cloned from rats, mice, cows
and humans and these NOSs have been used as the
major investigation models so far (16-21). A handful of
NOSs were cloned from other model organisms such
as insects (Drosophila melanogaster, (80-83)), amoeba
(Physarum polycephalum,(84, 85)) and mollusks (Limax
valentianus, (86)), but the number of reports on these
NOSs has remained negligible in contrast with the huge
literature on mammalian NOS (mNOS). With the rapid
evolution of the sequencing technologies, the presence
of NOS-like proteins has been identified in many other
genomes. Sequences homologous to NOS oxygenase
domain were found at first in various bacteria, from the
firmicutes and Deinococcus phyla: Bacillus subtilis,
Staphylococcus aureus, Deinococcus radiodurans
(2002, (73, 75, 76)) Bacillus Anthrax (2005, (87)). Later,
similar sequence blasts led to the identification of NOSs
in the genomes of bacteria from other phyla, such as
Actinobacteria (Streptomyces turgidiscabies, (88)) or
alpha-proteobacteria (Sorangium cellulosum, (89)).
Although these bacterial NOSs (bacNOS) corresponded
to a severely truncated form of the mammalian NOSs
(90, 91) (see Figure 3), this new family of protein was
spontaneously labelled bacterial NOS-like proteins
and used as models for mammalian NOS study, which
actually meant that they were believed to behave “like
mammalian NOS”.
3.2. Emergence of a new family of proteins
15 years later this reductive approach is no
longer tenable: today, a simple blast of the available
sequenced genomes led to the identification of
hundreds of new NOS-like proteins in reptiles,
turtles, amphibians, fishes, birds, insects, molluscs,
arthropods and various primitive metazoans such as
placozoan, amoeba, sponges, corals (see Tables). At
least one thousand NOSs were found in the sole eu­
bacteria kingdom (Table 1). However, the gene/protein
banks annotation has often proven to be misleading as
proteins were labelled as NOSs on the simple presence
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Figure 3. Bacterial NOS-like proteins have long been – and remain – considered as genuine NO° synthase that is involved in pathogenicity, virulence
and antibiotic resistance of pathogenic bacteria such as Staphylococcus aureus or Bacillus anthrax (161-163), in the biosynthesis of a phytotoxin by
Streptomyces turgidiscabies (78, 88) and in the metabolism reprogramming of Deinococcus radiodurans (164). Though, they harbor a completely
different structure from that of mammalian NOS as they only consist of a truncated oxygenase domain (between 300 and 350 residues). The absence
of any electron-purveying domain was circumvented by the call for external electron sources (162). However, this hypothesis overlooked the fact that
electron transfer has to be tightly orchestrated to allow for NO synthesis (31). This fine tuning is granted by the presence of the BH4 cofactor and by
the fusion of oxygenase and reductase domains within a single polypeptide. The absence of a CYPR domain in bacterial NOS prevents the control of
electron transfer rate. Likewise, the truncation of the N-terminal domain alters the binding of the BH4 cofactor, which is an indispensable element for NO°
production. Beyond these evident changes, bacterial oxygenase domain exhibits substitutions of several residues, and in particular a ValineàIsoleucine
switch that prevents NO° efficient release and tunes bacterial NOS into another enzymatic machinery. In this regard, a precise structural analysis of
oxygenase structure would have led to consider bacterial NOS as another type of NOS with singular (non-mammalian) structure and function.

of small regulatory or non-essential structural motifs (if
not linked to the presence of a small GTPase once
associated to NO synthase in plants or snails, (92,
93)). Using two different templates we have achieved
a blast against all available genomes and cleaned the
resulting protein list (see legend of Table 1) in order
to provide an update and accurate picture of this new
family of protein (Tables 1-3). We will here describe
the major phylogenetic groups where NOSs are to be
found but also those from where NOS is absent.
3.3. Prokaryotes, Eubacteria and Archae
In Prokaryotes. In the Eubacteria Domain,
NOSs were found in many phyla (Table 1): mostly
Firmicutes (>580 sequences), Actinobacteria (>320)
but also in Cyanobacteria (36), Deinococcus (20),
Proteobacteria (18), Bacteroidetes (11), Verrumicrobia
(10), Chloroflexi (2). Due to a bias in the choice of the
organisms to be sequenced, this list cannot reflect a
true distribution of NOSs in Eubacteria. However, it
shows how NOSs are spread throughout the bacterial
kingdom. NOSs are mostly found in the Terrabacteria
group. They are rarely found in Proteobacteria and
are absent from the genomes of other bacteria
(Acidobacteria, Aquificae, Chlorobi, Chlamydiae,
Fusobacteria, Spirochaetes, Thermotogae….).
Though, in the phyla where NOSs are highly present,
such as in Gram+ bacteria, NOSs are only present in
certain classes and absent from some major ones. This
is the case for Firmicutes: NOSs are present in Bacilli
but not in Clostridia. In the Bacilli class, NOSs are found
in all species of the Bacillales order but are completely
absent from the Lactobacillales order (except for the
unique Streptococcus pneumoniae). This patchiness
is well illustrated for Cyanobacteria: around 36 NOS
sequences (in species and subspecies) were found
(over at least 300 sequenced genomes) but the
rationale of their distribution does seem obvious. NOSs
are found throughout the cyanobacterial phyla (in
Nostocales, Oscillatoriales or Chroococales) in species
that correspond to different physiologies (subsections
1-5) or ecologies (fresh-water, marine, terrestrial…).
Which is highlighted in Firmicutes and Cyanobacteria
holds for the whole Eubacteria kingdom. The presence
of NOSs in only 13 distinct (alpha-, beta-, gamma- or
delta-) proteobacteria requires a combination of various
distinct explanations: loss from a common eubacterial
ancestor in this phyla and discrete horizontal genome
transfer (HGT) in very specific niches. As Hydrobacteria
and Terrabacteria might have diverged around three
billion years ago, implying a common ancestor long
before the Great Oxidation Event (GOE), a plural,
ramified and complex evolution scenario needs to
be found. The same heterogeneity is observed in the
Archae Domain. Only 10 sequences of NOS-related
proteins (over several hundreds of available genomes)
were found in species corresponding to only two orders
(Halobacteriales and Natrialbales) from the unique
Halobacteria class. Though, only a few archae species
from this very class exhibit a NOS-like sequence.
3.4. Eukaryotes: fungi and plants
In Eukaryotes: Fungi and Plants. The picture
remains much contrasted in the Eukaryote domain
(Table 2). NOSs seem to be absent from the Bikontes
group, (Excavata, Rhizaria, Alveolata) although it can
be found in two Stramenopiles species and in at least
two dozen of algae (94, 95). On the other hand, NOSs
seem more present in Unikontes, although only one
NOS-related sequence has been found in Amoebae

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Table 1. List of species that harbor NOS-like proteins in the Eubacteria and Archaea superkingdoms

Firmicutes-Bacilli-Bacillaceae-Bacillus
Bacillus subtilis Bacillus cihuensis Bacillus korlensis Bacillus pseudomycoides
Bacillus acidiceler Bacillus clarkii Bacillus krulwichiae Bacillus pumilus
Bacillus acidicola Bacillus clausii Bacillus lehensis G1 B. psychrosaccharolyticus
Bacillus aerophilus Bacillus coahuilensis Bacillus licheniformis Bacillus rhizosphaerae
Bacillus akibai Bacillus cohnii Bacillus ligniniphilus Bacillus safensis
Bacillus alcalophilus Bacillus cytotoxicus Bacillus luciferensis] Bacillus salsus
Bacillus alkalitelluris Bacillus daliensis Bacillus macauensis Bacillus selenitireducens
Bacillus altitudinis Bacillus eiseniae Bacillus malacitensis Bacillus shackletonii
Bacillus alveayuensis Bacillus encimensis Bacillus manliponensis Bacillus simplex
Bacillus aminovorans Bacillus enclensis Bacillus marisflavi Bacillus siamensis
B. amyloliquefaciens Bacillus endophyticus Bacillus marmarensis Bacillus sonorensis
Bacillus anthracis str. Bacillus farraginis B. massiliosenegalensis Bacillus tequilensis
Bacillus aquimaris Bacillus fastidiosus Bacillus massilioanorexius Bacillus testis
Bacillus aryabhattai Bacillus firmus Bacillus massiliogorillae Bacillus thuringiensis
Bacillus atrophaeus Bacillus flexus Bacillus megaterium Bacillus toyonensis
Bacillus aurantiacus Bacillus gaemokensis Bacillus mojavensis Bacillus trypoxylicola
Bacillus australimaris Bacillus ginsengihumi Bacillus muralis Bacillus vallismotis
Bacillus axarquiensis Bacillus gobiensis Bacillus nealsonii Bacillus vietnamensis
Bacillus badius Bacillus gottheilii Bacillus ndiopicus Bacillus velezensis
Bacillus beveridgei B. glycinifermentans Bacillus obstructivus Bacillus vireti
Bacillus bingmayongensis Bacillus halodurans Bacillus okhensis Bacillus weihaiensis
Bacillus bogoriensis Bacillus halotolerans Bacillus oleronius B. weihenstephanensis
Bacillus bombysepticus Bacillus horikoshii Bacillus mycoides Bacillus wiedmannii
Bacillus butanolivorans Bacillus indicus Bacillus panaciterrae Bacillus wakoensis
Bacillus chagannorensis Bacillus infantis Bacillus paralicheniformis Bacillus xiamenensis
Bacillus cellulosilyticus Bacillus lonarensis Bacillus patagoniensis Bacillus zhangzhouensis
Bacillus cellulasensis Bacillus isronensis Bacillus pseudofirmus Brevibacterium frigoritolerans
Bacillus cereus Bacillus koreensis Bacillus pseudalcaliphilus
Firmicutes-Bacilli-Bacillaceae-Lysinibacillus
Lysinibacillus massiliensis Lysinibacillus fusiformis ZC1 Lysinibacillus xyleni
Lysinibacillus sphaericus Lysinibacillus contaminans Lysinibacillus boronitolerans
Lysinibacillus acetophenoni Lysinibacillus manganicus Lysinibacillus varians
Lysinibacillus xylanilyticus Lysinibacillus sinduriensis Lysinibacillus saudimassiliensis
Firmicutes-Bacilli-Bacillaceae-Anoxybacillus
Anoxybacillus flavithermus Anoxybacillus aydernesis Anoxybacillus tepidamans
Anoxybacillus thermarum Anoxybacillus suryakudensis
Anoxybacillus amylolyticus Anoxybacillus pushchinoensis
Firmicutes-Bacilli-Bacillaceae-Pontibacillus
Pontibacillus marinus Pontibacillus yanchengensis Y32 Pontibacillus halophilus
Pontibacillus chungwhensis Pontibacillus litoralis
Firmicutes-Bacilli-Bacillaceae-Geobacillus
Geobacillus Stearothermophilus Geobacillus vulcani Geobacillus subterraneus
Geobacillus thermoleovorans Geobacillus jurassicus Geobacillus zalihae
Geobacillus kaustophilus Geobacillus thermocatenulatus Geobacillus uzenensis
Firmicutes-Bacilli-Bacillaceae-Oceanobacillus
Oceanobacillus picturae Oceanobacillus iheyensis Oceanobacillus jeddahense
Oceanobacillus oncorhynchi Oceanobacillus manasiensis Oceanobacillus timonensis
Oceanobacillus sojae Oceanobacillus massiliensis
Firmicutes-Bacilli-Bacillaceae-Halobacillus
Halobacillus aidingensis Halobacillus halophilus Halobacillus mangrovi
Halobacillus alkaliphilus Halobacillus karajensis Halobacillus massiliensis
Halobacillus dabanensis Halobacillus kuroshimensis Halobacillus salinus
Firmicutes-Bacilli-Bacillaceae
Anaerobacillus arseniciselenatis Terribacillus saccharophilus Domibacillus indicus
Anaerobacillus macyae Terribacillus halophilus Domibacillus aminovorans
Salsuginibacillus kocurii Terribacillus aidingensis Domibacillus iocasae
Salipaludibacillus aurantiacus Marinococcus halotolerans Domibacillus enclensis
Sediminibacillus halophilus Marinococcus luteus Virgibacillus proomii
Caldalkalibacillus thermarum Marinococcus halophilus Virgibacillus halodenitrificans
Aquibacillus sp. Fictibacillus phosphorivorans Virgibacillus pantothenticus
Edaphobacillus lindanitolerans Fictibacillus macauensis Virgibacillus chiguensis
Salimicrobium halophilum Fictibacillus arsenicus Virgibacillus dokdonensis
Salimicrobium jeotgali Paucisalibacillus globulus Psychrobacillus psychrotolerans
Salimicrobium album Thalassobacillus cyri Psychrobacillus psychrodurans

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Firmicutes-Bacilli-Paenibacillae
Brevibacillus brevis Brevibacillus choshinensis Saccharibacillus sacchari
Brevibacillus parabrevis Brevibacillus borstelensis Saccharibacillus kuerlensis
Brevibacillus formosus Brevibacillus panacihumi Cohnella thermotolerans
Brevibacillus borstelensis Brevibacillus reuszeri Cohnella laeviribosi
Firmicutes-Bacilli-Paenibacillae-Paenibacillus
Paenibacillus alginolyticus Paenibacillus elgii Paenibacillus peoriae
Paenibacillus algorifonticola Paenibacillus etheri Paenibacillus pini
Paenibacillus alvei Paenibacillus ferrarius Paenibacillus pinihumi
Paenibacillus amylolyticus Paenibacillus fonticola Paenibacillus polymyxa
Paenibacillus antarcticus Paenibacillus glacialis Paenibacillus popilliae
Paenibacillus assamensis Paenibacillus glucanolyticus Paenibacillus rhizosphaerae
Paenibacillaceae bacterium GAS479 Paenibacillus harenae Paenibacillus senegalensis
Paenibacillus beijingensis Paenibacillus gorillae Paenibacillus selenitireducens
Paenibacillus borealis Paenibacillus ihbetae Paenibacillus swuensis
Paenibacillus bovis Paenibacillus jamilae Paenibacillus taiwanensis
Paenibacillus camerounensis Paenibacillus kribbensis Paenibacillus terrae
Paenibacillus campinasensis Paenibacillus lactis Paenibacillus terrigena
Paenibacillus catalpae Paenibacillus lautus Paenibacillus thiaminolyticus
Paenibacillus chitinolyticus Paenibacillus larvae subsp. larvae Paenibacillus tianmuensis
Paenibacillus chondroitinus Paenibacillus macquariensis Paenibacillus taichungensis
Paenibacillus curdlanolyticus Paenibacillus massiliensis Paenibacillus typhae
Paenibacillus daejeonensis Paenibacillus naphthalenovorans Paenibacillus tyrfis
Paenibacillus dauci Paenibacillus odorifer Paenibacillus uliginis
Paenibacillus dendritiformis Paenibacillus pabuli Paenibacillus vortex
Paenibacillus ehimensis Paenibacillus panacisoli Paenibacillus xylanexedens
Paenibacillus pectinilyticus Paenibacillus yonginensis
Firmicutes-Bacilli-Alicyclobacillae
Alicyclobacillus acidocaldarius Alicyclobacillus contaminans Alicyclobacillus hesperidum
Alicyclobacillus acidiphilus Alicyclobacillus pomorum Alicyclobacillus acidoterrestris
Alicyclobacillus sendaiensis Alicyclobacillus vulcanalis
Alicyclobacillus mali Alicyclobacillus tengchongensis
Firmicutes-Bacilli-Planococcaceae
Sporosarcina globispora Jeotgalibacillus soli Bhargavaea cecembensis
Sporosarcina newyorkensis Jeotgalibacillus alimentarius Bhargavaea ginseng
Sporosarcina psychrophila Jeotgalibacillus campisalis Bhargavaea beijingensis
Sporosarcina ureae Jeotgalibacillus malaysiensis Rummeliibacillus stabekisii
Paenisporosarcina sp. Jeotgalicoccus psychrophilus Caryophanon tenue
Planomicrobium okeanokoites Planococcaceae bacterium VT-49 Caryophanon latum
Planomicrobium glaciei Solibacillus silvestris StLB046 Kurthia huakuii
Viridibacillus arvi Solibacillus kalamii Kurthia gibsonii
Viridibacillus arenosi Kurthia massiliensis
Firmicutes-Bacilli-Planococcaceae-Planococcus
Planococcus halocryophilus Planococcus antarcticus Planococcus massiliensis
Planococcus faecalis Planococcus donghaensis Planococcus rifietoensis
Planococcus maritimus Planococcus plakortidis
Firmicutes-Bacilli-Thermoactinomycetaceae
Shimazuella kribbensis Marininema halotolerans Risungbinella massiliensis
Thermoactinomyces vulgaris Marininema mesophilum
Firmicutes-Bacilli-Listeriaceae
Listeria grayi Listeria cornellensis Listeria weihenstephanensis
Listeria riparia Listeria grandensis Listeriaceae bacterium
Listeria newyorkensis Listeria rocourtiae Brochothrix campestris
Listeria booriae Brochothrix thermosphacta
Firmicutes-Bacilli-Staphylococcaceae
Staphylococcus arlettae Staphylococcus haemolyticus Staphylococcus schweitzeri
Staphylococcus agnetis Staphylococcus hominis Staphylococcus sciuri
Staphylococcus argenteus Staphylococcus hyicus Staphylococcus simiae
Staphylococcus aureus Staphylococcus intermedius Staphylococcus simulans
Staphylococcus caprae Staphylococcus lugdunensis Staphylococcus stepanovicii
Staphylococcus capitis Staphylococcus lutrae Staphylococcus warneri
Staphylococcus carnosus Staphylococcus microti Staphylococcus pettenkoferi
Staphylococcus cohnii Staphylococcus muscae Staphylococcus xylosus
Staphylococcus chromogenes Staphylococcus nepalensis Staphylococcus vitulinus
Staphylococcus delphini Staphylococcus pasteuri Staphylococcus succinus
Staphylococcus edaphicus Staphylococcus piscifermentans Macrococcus caseolyticus
Staphylococcus equorum Staphylococcus pseudintermedius Macrococcus canis
Staphylococcus epidermidis Staphylococcus massiliensis Macrococcus sp
Staphylococcus fleurettii Staphylococcus saprophyticus Nosocomiicoccus massiliensis
Staphylococcus gallinarum Staphylococcus schleiferi Nosocomiicoccus ampullae

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Firmicutes-Bacilli-Bacillales-unclassified
Acidibacillus ferrooxidans Exiguobacterium indicum Exiguobacterium oxidotolerans
Geomicrobium sp. JCM 19038 Exiguobacterium acetylicum Exiguobacterium chiriqhucha
Exiguobacterium undae Exiguobacterium aurantiacum Exiguobacterium mexicanum
Exiguobacterium enclense Exiguobacterium sibiricum 255-15 Exiguobacterium marinum
Exiguobacterium alkaliphilum Exiguobacterium antarcticum B7 Exiguobacterium sp.
Firmicutes-Bacilli-Lactobacillales-Streptococcaceae
Streptococcus pneumoniae
Actinobacteria-Actinomycetales-Pseudonocardiaceae-Pseudonocardia
Pseudonocardia dioxanivorans Pseudonocardia autotrophica Pseudonocardia oroxyli
Pseudonocardia ammonioxydans Pseudonocardia spinosispora Pseudonocardia thermophile
Pseudonocardia acaciae Pseudonocardia sp.
Actinobacteria-Actinomycetales-Pseudonocardiaceae-Amycolatopsis
Amycolatopsis mediterranei U32 Amycolatopsis vancoresmycina Amycolatopsis pretoriensis
Amycolatopsis azurea DSM 43854 Amycolatopsis lurida NRRL 2430 Amycolatopsis regifaucium
Amycolatopsis orientalis Amycolatopsis lexingtonensis Amycolatopsis lurida
Amycolatopsis japonica Amycolatopsis rifamycinica Amycolatopsis coloradensis
Amycolatopsis alba Amycolatopsis balhimycina Amycolatopsis australiensis
Amycolatopsis decaplanina Amycolatopsis tolypomycina Amycolatopsis saalfeldensis
Amycolatopsis keratiniphila Amycolatopsis xylanica Amycolatopsis sp.
Actinobacteria-Actinomycetales-Pseudonocardiaceae
Saccharopolyspora erythraea NRRL Actinoalloteichus cyanogriseus Lechevalieria aerocolonigenes
Saccharopolyspora sp(i)a NRRL Actinoalloteichus spitiensis Lechevalieria xinjiangensi
Saccharopolyspora spinosa Actinoalloteichus sp. Lechevalieria fradiae
Saccharopolyspora hirsuta Actinoalloteichus hymeniacidonis Allokutzneria albata
Saccharopolyspora flava Actinokineospora inagensis Allokutzneria sp. NRRL B-24872
Saccharopolyspora antimicrobica Actinokineospora bangkokensis Kutzneria albida
Saccharopolyspora shandongensis Actinokineospora terrae Kutzneria sp. 744
Saccharomonospora xinjiangensis Actinokineospora enzanensis Catenulispora acidiphila
Saccharothrix espanaensis A. spheciospongiae Crossiella equi
Saccharothrix syringae Actinosynnema mirum Lentzea albida
Saccharothrix sp. Actinosynnema pretiosum Lentzea albidocapillata
Saccharothrix sp. Actinophytocola xanthii Lentzea kentuckyensis
Saccharothrix sp. Alloactinosynnema album Lentzea flaviverrucosa
Kibdelosporangium phytohabitans Alloactinosynnema iranicum Lentzea guizhouensis
Kibdelosporangium aridum Alloactinosynnema sp. Lentzea jiangxiensis
Kibdelosporangium sp. Actinosynnema sp. Lentzea violacea
Actinomycetospora chiangmaiensis Actinophytocola xinjiangensis Lentzea waywayandensis
Actinobacteria-Actinomycetales-Micromonosporaceae-Micronospora
Micromonospora carbonacea Micromonospora peucetia Micromonospora Micromonospora coxensis
Micromonospora globosa rosaria Micromonospora nigra
Micromonospora matsumotoense Micromonospora rifamycinica M. purpureochromogenes
Micromonospora echinofusca Micromonospora siamensis Micromonospora globosa
Micromonospora mirobrigensis Micromonospora yangpuensis Micromonospora pattaloongensis
Actinobacteria-Actinomycetales-Micromonosporaceae
Actinoplanes missouriensis Salinispora arenicola Hamadaea tsunoensis
Actinoplanes utahensis Salinispora pacifica Catelliglobosispora koreensis
Actinoplanes globisporus Salinispora tropica Couchioplanes caeruleus
Actinoplanes subtropicus Actinoplanes atraurantiacus Hamadaea tsunoensis
Actinoplanes friuliensis Actinoplanes rectilineatus Asanoa ishikariensis
Actinoplanes philippinensis Actinoplanes sp. Asanoa hainanensis
Actinoplanes derwentensis Catenuloplanes japonicus Verrucosispora sp.
Actinoplanes awajinensis Dactylosporangium aurantiacum
Actinobacteria-Actinomycetales-Streptomycetaceae
Streptacidiphilus jeojiense Streptacidiphilus melanogenes Streptoalloteichus hindustanus
Streptacidiphilus albus Streptacidiphilus neutrinimicus Kitasatospora mediocidica
Streptacidiphilus jiangxiensis Streptacidiphilus anmyonensis Kitasatospora azatica
Streptacidiphilus carbonis Streptomycetaceae bacterium

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Actinobacteria-Actinomycetales-Streptomycetaceae-Streptomyces
Streptomyces scabiei 87.22 Streptomyces qinglanensis Streptomyces rubidus
Streptomyces alboverticillatus Streptomyces ipomoeae Streptomyces rubellomurinus
Streptomyces abyssalis Streptomyces katrae Streptomyces turgidiscabies
Streptomyces acidiscabies 84-104 Streptomyces lavendulae Streptomyces uncialis
Streptomyces alboviridis Streptomyces mirabilis Streptomyces virginiae
Streptomyces alni Streptomyces globisporus Streptomyces viridochromogenes
Streptomyces aureofaciens Streptomyces griseoluteus Streptomyces vietnamensis
Streptomyces avermitilis MA-4680 Streptomyces griseochromogenes Streptomyces scabrisporus
Streptomyces avicenniae Streptomyces harbinensis Streptomyces specialis
Streptomyces bikiniensis Streptomyces indicus Streptomyces viridifaciens
Streptomyces diastatochromogenes Streptomyces nanshensis Streptomyces venezuelae
Streptomyces erythrochromogenes Streptomyces olivochromogenes Streptomyces wedmorensis
Streptomyces exfoliatus Streptomyces pathocidini Streptomyces xanthophaeus
Streptomyces flavochromogenes Streptomyces puniciscabiei Streptomyces yanglinensis
Streptomyces fulvoviolaceus Streptomyces purpeofuscus Streptomyces yokosukanensis
Streptomyces glaucescens Streptomyces roseus Streptomyces zhaozhouensis
Streptomyces glauciniger Streptomyces roseoverticillatus
Streptomyces griseus
Actinobacteria-Actinomycetales-Nocardiaceae-Nocardia
Nocardia amamiensis Nocardia beijingensis Nocardia salmonicida
Nocardia asiatica Nocardia exalbida Nocardia soli
Nocardia altamirensis Nocardia gamkensis Nocardia thailandica
Nocardia asteroides NBRC Nocardia lijiangensis Nocardia transvalensis
Nocardia abscessus Nocardia harenae Nocardia takedensis
Nocardia arthritidis Nocardia crassostreae Nocardia tenerifensis
Nocardia anaemiae Nocardia alba Nocardia terpenica
Nocardia acidivorans Nocardia niwae Nocardia vulneris
Nocardia arizonensis Nocardia otitidiscaviarum Nocardia vinacea
Nocardia brasiliensis Nocardia pseudovaccinii Nocardia xishanensis
Nocardia amikacinitolerans Nocardia pseudobrasiliensis Nocardia yamanashiensis
Nocardia brevicatena Nocardia puris
Nocardia araoensis Nocardia mexicana
Actinobacteria-Actinomycetales-Nocardiaceae-Rhodococcus
Rhodococcus opacus B4/ M213 Rhodococcus rhodnii Rhodococcus kyotonensis
Rhodococcus imtechensis RKJ300 Rhodococcus fascians Rhodococcus marinonascens
Rhodococcus jostii RHA1 Rhodococcus rhodochrous Rhodococcus maanshanensis
Rhodococcus erythropolis Rhodococcus qingshengii Rhodococcus tukisamuensis
Rhodococcus enclensis Rhodococcus yunnanensis
Rhodococcus wratislaviensis Rhodococcus koreensis
Actinobacteria-Actinomycetales-Streptosporangiaceae
Streptosporangium roseum DSM Nonomuraea coxensis Microbispora rosea
Streptosporangium amethystogenes Nonomuraea candida Herbidospora yilanensis
Streptosporangium canum Nonomuraea solani Microtetraspora glauca
Streptosporangium amethystogenes Nonomuraea coxensis Planobispora rosea
Streptosporangium sp. Nonomuraea wenchangensis Planomonospora sphaerica
Herbidospora cretacea Nonomuraea jiangxiensis Sinosporangium album
Herbidospora daliensis Nonomuraea maritima
Herbidospora sakaeratensis Herbidospora mongoliensis
Other Actinobacteria
Actinobacteria-Corynebacteriales Actinobacteria-Streptosporangiales
Skermania piniformis Actinomadura flavalba
Mycobacterium abscessus. Actinomadura hibisca
Corynebacterium sp. OG2 Actinomadura formosensis
Actinobacteria-Micrococcales Streptosporangium subroseum
Brachybacterium. faecium Actinobacteria-Jiangellales
Actinobacteria-Propionobacteriales Jiangella gansuensis
Kribbella catacumbae Jiangella alkaliphila
Kribbella sp. ALI-6-A Jiangella muralis
Actinobacteria-Frankiales Jiangella alba
Cryptosporangium arvum Unclassified Actinobacteria
Sporichthya polymorpha Actinobacteria bacterium OV450
Actinobacteria-Nakamurellales
Nakamurella multipartita DSM
Cyanobacteria
Aphanocapsa montana Aliterella atlantica CENA595 Microcoleus sp
Scytonema hofmanni Neosynechococcus sphagnicola Microcoleus vaginatus FGP-2
Aphanizomenon flos-aquae Nostoc sp Crinalium epipsamum PCC9333
Dolichospermum circinale Anabaena sp Planktothrix sp.
Calothrix sp PCC 7103 Chlorogloeopsis fritschii Planktothrix paucivesiculata PCC9631
Microchaete sp PCC Mastigocoleus testarum Synechococcus sp

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Bacteroidetes
Marivirga sericea B Spirosoma montaniterrae Spirosoma linguale
Phaeodactylibacter xiamenensis Spirosoma radiotolerans Spirosoma rigui
Algoriphagus sp. M8-2 Spirosoma fluviale Spirosoma sp.
Bacteroidetes bacterium MED-G21
Deinococcus
Deinococcus maricopensis Deinococcus aquatilis Deinococcus radiodurans
Deinococcus geothermalis Deinococcus gobiensis Deinococcus murrayi
Deinococcus peraridilitoris Deinococcus deserti VCD115 Deinococcus pimensis
Deinococcus pimensis Deinococcus proteolyticus MRP Deinococcus grandis
Deinococcus sp. Deinococcus misasensis Deinococcus reticulitermitis
Deinococcus actinosclerus Deinococcus wulumuqiensis
Proteobacteria
Proteobacteria (alpha)-Rhodobacterales Proteobacteria (gamma)-Enterobacterales
Silicibacter sp. TrichCH4B Klebsiella pneumoniae
Jannaschia seosinensis Proteobacteria (beta) -Burkholderiales Hydrogenophaga sp. A37
Henriciella sp. Proteobacteria (alpha)-Sphingomonadales
Pseudooceanicola sp. Erythrobacter sp.
Pseudooceanicola marinus Sphingomonas elodea
Pseudooceanicola antarticus Sphingomonas pituitosa
Proteobacteria (alpha)-Rhodospirillales Sphingomonas sp. Leaf
Rhodospirillaceae bacterium TMED140 Novosphingobium subterraneum :
Caenispirillum bisanense Alphaproteobacteria bacterium TMED87 PA4
Proteobacteria (alpha)-Sneathiellales Proteobacteria (delta)-Myxococcales
Sneathiella sp. Sorangium cellulosum
Verrucomicrobia
Verrucomicrobia bacterium SCGC AAA168-F10 Opitutae bacterium TMED149
Verrucomicrobia subdivision 6 bacterium BACL9 Roseibacillus sp
Verrucomicrobia bacterium SCGC AAA168F10 Rubritalea squalenifaciens DSM 18772
Verrucomicrobiaceae bacterium TMED137
Verrucomicrobiaceae bacterium TMED76
Verrucomicrobiaceae bacterium TMED86
Other Eubacteria
Chloroflexi Balneolaeota
Ktedonobacter racemifer Balneola vulgaris
Coraliomargarita sp. TMED73 Balneolaceae bacterium TMED105
Archaea-Euryarchaeota-Halobacteria-Natrialbales
Natronobacterium gregoryi SP2 Halobiforma nitratireducens JCM 10879
Natronobacterium texcoconense Halostagnicola kamekurae
Natrialbaceae archaeon Halostagnicola sp.
Archaea-Euryarchaeota-Halobacteria-Halobacteriales
Natronomonas pharaonis Halovenus aranensis
Halorientalis sp. IM1011 Halalkalicoccus paucihalophilus
Census has been achieved using the online NCBI Blastp suite ©. Two distinct Query sequences have been used (bsNOS from Bacillus subtilis subsp.
spizizenii TU-B-10, and iNOSoxy ref) using BLOSUM 62 as matrix and low Gap Costs (Existence 6, Extension 2). Validity of retrieved sequences was
verified via the presence of the NOS-specific heme binding WRNxxxC motif. The list was updated on the 23rd of November 2017. Used nomenclature
is derived from the NCBI version of Lifemap ©

Table 2. List of NOS-like proteins in non-metazoan Eukaryotes

Viridiplantae

Klebsormidium nitens Bathycoccus prasinos Helicodictyon planctonicum

Klebsormidium flaccidum Gonium pectorale Leptosira obovata
Chaetosphaeridium globosum Bolbocoleon piliferum Pandorina morum
Cosmarium subtumidum Volvox aureus Nephroselmis pyriformis
Planophila terrestris Pleurastrum insigne Pteromonas sp
Chlamydomonas cribrum Golenkinia longispicula Ostreococcus tauri
Phacotus lenticularis Scherffelia dubia Ostreococcus lucimarinus

Stramenopiles-Bacillariophyta-Coscinodiscophyceae-Thalassiosirales

Thalassiosira oceanica

Stramenopiles-Oomycetes-Saprolegniales-Saprolegniaceae

Thraustotheca clavata

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Opisthokonta-Fungi-Dikarya-Ascomycota

Aspergillus arachidicola Colletotrichum chlorophyti Glomerella graminicola M1.001

Aspergillus bombycis Colletotrichum fioriniae PJ7 Lepidopterella palustris CBS 45
Aspergillus carbonarius ITEM Colletotrichum graminicola M1.001 Macrophomina phaseolina
Aspergillus kawachii Colletotrichum higginsianum Neofusicoccum parvum UCRNP2
Aspergillus luchuensis Colletotrichum gloeosporioides Oidiodendron maius Zn
Aspergillus nomius Colletotrichum incanum] Ophiocordyceps sinensis CO18
Aspergillus oryzae Colletotrichum orbiculare MAFF 240422 Ophiocordyceps unilateralis
Aspergillus niger Colletotrichum orchidophilum Phialocephala scopiformis
Aspergillus flavus Colletotrichum salicis Phialocephala subalpina
Aspergillus parasiticus SU-1 Colletotrichum simmondsii Pseudomassariella vexata
Aspergillus welwitschiae Colletotrichum sublineola Umbilicaria pustulata
Dothistroma septosporum Colletotrichum tofieldiae Verruconis gallopava
Elaphomyces granulatus Coniosporium apollinis CBS 100218 Zymoseptoria tritici IPO323
Endocarpon pusillum Z07020 Coniochaeta ligniaria NRRL 30616 Zymoseptoria brevis

Opisthokonta-Fungi-Dikarya-Basidiomycota

Rhizoctonia solani

Opisthokonta-Fungi-Chytridiomycota

Gonapodya prolifera JEL478

Opisthokonta-Opisthokonta incertae sedis-Ichthyosporea-Ichthyophonida

Sphaeroforma arctica JP610

Amoebozoa

Physarum polycephalum

Heterolobosea

Naegleria gruberi strain NEG-M

Haptophyceae-Prymnesiales-Chrysochromulinaceae

Chrysochromulina sp. CCMP291

The same protocol as in Table 1 was used. Additional species in viridiplantae come from Jeandroz et al. (94).

Table 3. List of NOS-like proteins in Metazoa

Placozoa

Trichoplax adhaerens

Porifera

Amphimedon queenslandica

Cnidaria-Anthozoa

Nematostella vectensis Discosoma striata Stylophora pistillata

Acropora digitifera Orbicella faveolata Exaiptasia pallida

Cnidaria-Hydrozoa

Hydra magnipapillata Hydra vulgaris

Lophotrochozoa-Brachyopoda

Lingula anatina

Lophotrochozoa-Bryozoa

Bugula neritina

Lophotrochozoa-Annelida

Capitella teleta

Lophotrochozoa-Mollusca-Bivalvia

Crassostrea gigas Azumapecten farreri Mytilus galloprovincialis

Crassostrea virginica Mizuhopecten yessoensis

Lophotrochozoa-Mollusca

Sepia officinalis Octopus bimaculoides

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Lophotrochozoa-Mollusca-Gastropoda

Aplysia californica Lottia gigantea Planorbella trivolvis

Ambigolimax valentianus Lymnaea stagnalis Stramonita canicula 
Biomphalaria glabrata Limulus polyphemus Stramonita haemastoma
Haliotis asinina

Ecdysozoa-Panarthropoda-Tardigrada

Hypsibius dujardini Ramazzottius varieornatus

Ecdysozoa-Arthropoda-Chelicerata-Arachnida

Ixodes scapularis Parasteatoda tepidariorum Sarcoptes scabiei

Stegodyphus mimosarum Tetranychus urticae Euroglyphus maynei

Ecdysozoa-Arthropoda-Crustacea-Maxillopoda

Tigriopus japonicus

Ecdysozoa-Arthropoda-Crustacea-Branchiopoda

Daphnia pulex Daphnia magna

Ecdysozoa-Arthropoda-Crustacea-Eumalacostraca-Amphipoda

Hyalella azteca

Ecdysozoa-Arthropoda-Crustacea-Eumalacostraca-Decapoda

Panulirus argus Marsupenaeus japonicus Litopenaeus vannamei

Carcinus maenas Fenneropenaeus chinensis Scylla paramamosain
Gecarcinus lateralis Penaeus monodon Portunus trituberculatus

Ecdysozoa-Arthropoda-Hexapoda-Collembola

Orchesella cincta Folsomia candida

Ecdysozoa-Arthropoda-Hexapoda-Insecta-Diptera

Anopheles darlingi Drosophila mojavensis Drosophila biarmipes

Anopheles dirus Drosophila grimshawi Drosophila takahashii
Anopheles gambiae Drosophila simulans Drosophila elegans
Anopheles stephensi Drosophila willistoni Drosophila serrata
Anopheles sinensis Drosophila ananassae Drosophila suzukii
Aedes aegypti Drosophila melanogaster Drosophila kikkawai
Aedes albopictus Drosophila persimilis Drosophila bipectinata
Bactrocera dorsalis Drosophila pseudoobscura Drosophila arizonae
Bactrocera cucurbitae Drosophila eugracilis Drosophila rhopaloa
Bactrocera latifrons Drosophila ficusphila Drosophila busckii
Bactrocera oleae Drosophila miranda Lucilia cuprina
Ceratitis capitata Drosophila virilis Musca domestica
Clunio marinus Drosophila obscura Rhagoletis zephyria
Drosophila yakuba Drosophila sechellia Stomoxys calcitrans
Drosophila erecta Zeugodacus cucurbitae

Ecdysozoa-Arthropoda-Hexapoda-Insecta-Hemiptera

Acyrthosiphon pisum Bemisia tabaci Rhodnius prolixus

Cimex lectularius Halyomorpha halys Nilaparvata lugens
Diuraphis noxia Diaphorina citri Myzus persicae

Ecdysozoa-Arthropoda-exapoda-Insecta-Coleoptera

Aethina tumida Luciola cruciate Nicrophorus vespilloides

Dendroctonus ponderosae Luciola lateralis Tribolium castaneum
Aquatica lateralis Agrilus planipennis Lucidina biplagiata
Oryctes borbonicus Anoplophora glabripennis

Ecdysozoa-Arthropoda-Hexapoda-Insecta-Lepidopterea

Amyelois transitella Manduca sexta Papilio Xuthus

Danaus plexippus Bombyx mori Papilio polytes
Mythimna separate Helicoverpa armigera Papilio machaon
Plutella xylostella Operophtera brumata Papilio polytes
Pieris rapae Spodoptera exigua

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Ecdysozoa-Arthropoda-Hexapoda-Insecta-Hymenoptera

Acromyrmex echinatior Copidosoma floridanum Nasonia vitripennis

Apis cerana (cerana) Cyphomyrmex costatus Neodiprion lecontei
Apis mellifera Eufriesea Mexicana Orussus abietinus
Apis florea Fopius arisanus Polistes dominula
Apis dorsata Diachasma alloeum Polistes canadensis
Athalia rosae Dinoponera quadriceps Philanthus triangulum
Atta colombica Dufourea novaeangliae Solenopsis invicta
Atta cephalotes Harpegnathos saltator Pogonomyrmex barbatus
Bombus terrestris Habropoda laboriosa Pseudomyrmex gracilis
Bombus impatiens Lasius niger Trachymyrmex cornetzi
Camponotus floridanus Linepithema humile Trachymyrmex septentrionalis
Cephus cinctus Megachile rotundata Trichogramma pretiosum
Cerapachys biroi Melipona quadrifasciata Vollenhovia emeryi
Ceratina calcarata Microplitis demolitor Wasmannia auropunctata
Ceratosolen solmsi marchali Monomorium pharaonis

Ecdysozoa-Arthropoda-Hexapoda-Insecta-Orthoptera

Gryllus bimaculatus Acheta domesticus

Ecdysozoa-Arthropoda-Hexapoda-Insecta-Phthiraptera

Pediculus humanus corporis

Ecdysozoa-Arthropoda- Hexapoda-Insecta-Blattodea

Zootermopsis nevadensis

Echinodermata

Acanthaster planci Strongylocentrotus purpuratus Apostichopus japonicus

Cephalochordata

Branchiostoma floridae Branchiostoma belcheri

Tunicata

Ciona intestinalis

Craniata-Actinopterygii-Chondrichthyes

Callorhinchus milii Scyliorhinus canicula Rhincodon typus

Craniata-Actinopterygii-Clupeocephala

Astyanax mexicanus Ictalurus punctatus Pundamilia nyererei

Acanthochromis polyacanthus Kryptolebias marmoratus Poecilia formosa
Austrofundulus limnaeus Labrus bergylta Poecilia latipinna
Boleophthalmus pectinirostris Larimichthys crocea Poecilia mexicana
Clarias sp. SM-2014 Lates calcarifer Poecilia reticulata
Clarias batrachus Megalobrama amblycephala Pygocentrus nattereri
Carassius auratus Maylandia zebra Salmo salar
Carassius carassius Micropogonias undulatus Sciaenops ocellatus
Clupea harengus Monopterus albus Seriola dumerili
Ctenopharyngodon idella Neolamprologus brichardi Scophthalmus maximus
Cyprinodon variegatus Nothobranchius furzeri Stegastes partitus
Cyprinus carpio Notothenia coriiceps Sinocyclocheilus rhinocerous
Cynoglossus semilaevis Oreochromis niloticus Sinocyclocheilus grahami
Danio rerio Oncorhynchus mykiss Sinocyclocheilus anshuiensis
Esox lucius Oncorhynchus kisutch Tetraodon nigroviridis
Fundulus heteroclitus Oryzias latipes Takifugu poecilonotus
Hippocampus kuda Paralichthys olivaceus Takifugu rubripes
Hippocampus comes Platichthys flesus Xiphophorus maculatus
Haplochromis burtoni

Craniata-Actinopterygii-Semionotiformes

Lepisosteus oculatus

Craniata-Actinopterygii-Osteoglossiformes

Scleropages formosus

Latimeria chalumnae

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Craniata-Sarcopterygii-Tetrapoda-Sauria-Aves

Acanthisitta chloris Columba livia Corvus brachyrhynchos Mesitornis unicolor

Amazona aestiva] Corvus cornix cornix Nestor notabilis
Anas platyrhynchos Colius striatus Nipponia nippon
Anser sp. Coturnix japonica Numida meleagris
Anser anser Cuculus canorus Opisthocomus hoazin
Anser cygnoides domesticus Egretta garzetta Parus major
Antrostomus carolinensis Eurypyga helias, Patagioenas fasciata monilis
Apaloderma vittatum Falco peregrinus Pelecanus crispus
Aptenodytes forsteri Falco cherrug Phaethon lepturus
Apteryx australis mantelli Ficedula albicollis Phalacrocorax carbo
Aquila chrysaetos canadensis Fulmarus glacialis Phoenicopterus ruber ruber
Balearica regulorum gibbericeps Gallus gallus Picoides pubescens
Buceros rhinoceros silvestris Gavia stellata Podiceps cristatus
Calidris pugnax Geospiza fortis Pseudopodoces humilis
Callipepla squamata Haliaeetus leucocephalus Pterocles gutturalis
Calypte anna [Haliaeetus albicilla Pygoscelis adeliae
Caprimulgus carolinensis Leptosomus discolor Serinus canaria
Cariama cristata Lepidothrix coronate Sturnus vulgaris
Cathartes aura Lonchura striata domestica Struthio camelus australis
Chaetura pelagica Manacus vitellinus Taeniopygia guttata
Charadrius vociferus Meleagris gallopavo Tauraco erythrolophus
Chlamydotis macqueenii Melopsittacus undulatus Tinamus guttatus
Colinus virginianus Merops nubicus Tyto alba
Colius striatus Zonotrichia albicollis

Craniata-Sarcopterygii-Tetrapoda-Sauria-Crocodylia

Alligator mississippiensis Crocodylus porosus

Alligator sinensis Gavialis gangeticus

Craniata-Sarcopterygii-Tetrapoda-Sauria-Lepidosaure

Anolis carolinensis Ophiophagus hannah Pogona vitticeps

Aspidoscelis uniparens Protobothrops mucrosquamatus Phrynocephalus przewalskii
Python bivittatus Gekko japonicus Phrynocephalus erythrurus Thamnophis
Phrynocephalus erythrurus sirtalis

Craniata-Sarcopterygii-Tetrapoda-Sauria-Testudines

Chrysemys picta bellii Pelodiscus sinensis Chelonia mydas

Craniata-Sarcopterygii-Tetrapoda-Amphibia

Xenopus (Silurana) tropicalis Rhinella marina

Xenopus laevis Nanorana parkeri

Craniata-Sarcopterygii-Tetrapoda-Metatheria

Monodelphis domestica Phascolarctos cinereus Sarcophilus harrisii

Craniata-Sarcopterygii-Tetrapoda-Prototheria

Ornithorhynchus anatinus

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Xenarthra

Dasypus novemcinctus

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Glires-Rodentia

Castor canadensis Ictidomys tridecemlineatus Mus caroli

Cavia porcellus Jaculus jaculus Nannospalax galili
Chinchilla lanigera Marmota marmota marmota Octodon degus
Cricetulus griseus Mesocricetus auratus Neotoma lepida
Dipodomys ordii Meriones unguiculatus Peromyscus maniculatus bairdii
Eospalax fontanierii baileyi Microtus ochrogaster Rattus rattus
Fukomys damarensis Mus musculus Rattus norvegicus
Heterocephalus glaber Mus pahari

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Glires-Lagomorphes

Ochotona princeps Ochotona curzoniae

Oryctolagus cuniculus Ochotona collaris

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Ruminants

Bison bison bison Bos mutus Odocoileus virginianus texanus

Bos taurus Bubalus bubalis Ovis aries
Bos bovis Cervus elaphus hippelaphus Ovis aries musimon
Bos grunniens mutus Ophiophagus hannah] Pantholops hodgsonii
Bos indicus Capra hircus

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Revisiting the structure, function and evolution of NOS family

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Insectivores

Condylura cristata Sorex araneus Erinaceus europaeus

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Carnivores

Acinonyx jubatus Felis catus Ursus maritimus

Ailuropoda melanoleuca Mustela putorius furo Acinonyx jubatus
Canis lupus familiaris Panthera tigris altaica Leptonychotes weddellii Enhydra lutris
Panthera pardus Odobenus rosmarus divergens kenyoni
Neomonachus schauinslandi

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Cetartiodactyla

Physeter catodon Balaenoptera acutorostrata scammoni Sus scrofa

Delphinapterus leucas Camelus ferus Lipotes vexillifer
Camelus dromedaries Camelus bactrianus Orcinus orca
Vicugna pacos Tursiops truncatus

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Perissodactyles

Equus caballus Ceratotherium simum simum

Equus przewalskii Cheval Equus asinus

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Chiropteres

Pteropus alecto Rousettus aegyptiacus Eptesicus fuscus

Hipposideros armiger Pteropus vampyrus Myotis lucifugus Rhinolophus sinicus
Miniopterus natalensis Myotis davidii

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Dermopteres

Galeopterus variegatus Chrysochloris asiatica

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Pholidota

Loxodonta Africana Elephantulus edwardii Tupaia chinensis

Echinops telfairi Orycteropus afer afer Manis javanica

Craniata-Sarcopterygii-Tetrapoda-Eutheria-Primates

Aotus nancymaae Homo sapiens Pan troglodytes

Callithrix jacchus Macaca mulatta Pongo abelii
Carlito syrichta Macaca fascicularis Pan paniscus
Cebus capucinus imitator Macaca nemestrina] Papio Anubis
Cercocebus atys Mandrillus leucophaeus Propithecus coquereli
Chlorocebus sabaeus Microcebus murinus Rhinopithecus roxellana
Colobus angolensis palliatus Nomascus leucogenys Rhinopithecus bieti
Gorilla gorilla gorilla Otolemur garnettii Saimiri boliviensis

Same protocol as in Table 1. Species might include various isoforms.

and none in Choenaflagellata. Fungal NOSs were
only found in the Ascomycota phylum (38 species)
with the intriguing presence of NOS sequence in
one Basidiomycota and one Chytridiomycota fungi,
and no related sequences in Glomeromycota nor in
Zygomycota. NOS sequences were clustered in only 4
classes and 13 different orders of fungi (among dozens)
belonging to the same Pezizomycotina subphylum.
However, the absence of NOSs in Saccharomycotina
yeasts and in most of the sequenced species of these
classes (Penicillium, Microsporum, Blastomyces and
etc.) highlight the same patchy distribution of NOS in
the fungal tree of life (Figure 4A). The pattern in the
Green lineage is more puzzling: whereas land plants
ubiquitously uses NO° for a wide range of purposes
as different as immunity, stress response, growth or
mycorrhizal symbiosis regulation ((96-100), no NOS
sequence is to be found in land plants, which suggest
that they might have lost their NO-Synthase in the
course of evolution. NOS-related proteins today can
only be found in a discrete number of green algae
from both Chlorophytes and Streptophytes phyla (94,
95). Though, NOSs are not ubiquitous in green algae
since only 23 NOSs were identified among the several
dozens of algal genomes that have been sequenced
so far. For example, NOS seems omnipresent in
the Bathycoccaceae family but is absent from the
Mamiellaceae family that yet belongs to the same
Mamiellales order. Likewise, many NOSs sequences
have been found in the Chlamydomonadales order
although NOS is clearly absent from the model alga
Chlamydomonas reinhardtii (Figure 4B). Once again,
the distribution of NOSs found in algae and fungi
remains apparently inexplicable.
3.5. Metazoan
A few articles have reviewed in the past years
the presence of NOSs in metazoan and in particular in
invertebrate and marine organisms (101-103). However,
the number of sequenced genomes has exponentially
increased since then, giving rise to a wealth of new
data that we will summarize and analyze here (Table
3). NOSs are found in Porifera and in all radiates phyla

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Revisiting the structure, function and evolution of NOS family
Figure 4. Schematic patterns of NOS distribution in three representative clades. Species phylogeny was drawn from NCBI and (165). Green stars that
NOS-like proteins have been found in (almost) all species of the branch. Red sign means that no homologous sequences were found in the branch. A.
Fungi. B. Algae. C. Basal metazoans.
such as Cnidaria and Placozoa. In Cnidaria, NOSs are
mostly found in Anthozoan. NOSs are absent from the
sequenced genomes of Myxozoan. NOSs have not
yet been identified in jellyfishes or seawasps, but they
are not completely absent from the Medusozoa family
as NOSs have been found in Hydra vulgaris (104).
Besides, NOSs are not ubiquitous in Anthozoan and
seem absent from Octocorallia species. Once again,
the distribution of NOS in the Cnidaria Phylum does
not seem homogeneous (Figure 4C). This patchy
distribution is still observed in Bilateria and in particular
in Protostomes. NOSs are absent from major worm’s
phyla (Platyhelminthes, Acanthocephala, Rotifera,
Nemertea, Nematodes, Gastrotriches…), with only
one NOS in the genome of one annelid worm (Capitella
teleta). In Ecdysozoan, NOSs are found in arthropods
and tardigrades. In Lophotrochozoan, NOSs are
primarily found in mollusks in, with singlet presence in
Bryozoa and Brachyopods (Figure 4B). NOS presence
becomes ubiquitous only at the level of Chordata: the
genome of all Chordata species seems to harbor a
NOS-related sequence so far.
4. A NEW AND HETEROGENEOUS FAMILY
OF PROTEINES
4.1. The impasse of standard phylogenetic
analysis
This long “topological” list of NOSs (that
remains to be frequently updated) conveys three
148
major conclusions: i) this new, large and diverse family
of proteins calls for a thorough characterization; ii) the
considerable number of NOS proteins throughout the
tree of life makes impossible the systematic structural
and functional characterization of each of these
NOSs; iii) mammalian NOSs do not represent the
most important class of species that harbor a NOS in
their genome and as such mNOS can no longer be
considered as the archetypal NOS. It is tempting to
achieve a phylogenetic analysis of this large population
of proteins in order to see if some rational evolutive story
can emerge from their patchy distribution, as it has been
done many times in the past (102). We achieved such
an analysis by using a careful sampling of NOSs that
would be representative of the heterogeneity of NOS
distribution: we selected the sequence of 93 different
NOSs from various prokaryotes and eukaryotes. The
multiple-alignment of these sequences was used to
generate a phylogenetic tree (Figure 5, see legend).
The major analyses of this tree are reported in Figure
6. It seems that this tree can be decomposed in
distinct parts that each display different patterns. In
vertebrates, NOSs phylogeny seems to unfold along
the type of the isoforms (eNOS, nNOS, iNOS). This is
not the case for the other metazoans and in particular
for invertebrate’s NOSs that seem to follow a species-
based phylogeny. In Eukaryotes (Plants, Fungi,
Stramenopiles…), the phylogeny seems more related
to ecological/physiological factors: the NOS from the
photosynthetic Stramenopiles is on the same branch as
algae’s NOSs, whereas the NOS from the oomycetes
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Revisiting the structure, function and evolution of NOS family

Figure 5. Tentative phylogenetic tree of NOS protein family. Phylogenetic tree of a selection of 93 NOSs representative of NOS phyletic diversity,
including NOSs from Cyanobacteria (Scytonema hofmanni, Crinalium epipsammum, Anabaenopsis circularis, Neosynechococcus sphagnicola, Nostoc
linckia, Nostoc sp. PCC 7107, Mastigocoleus testarum, Calothrix sp. NIES-2100), Firmicutes (Anaerobacillus arseniciselenatis, Bacillus subtilis,
streptococcus pneumoniae), Actinobacteria (Streptomyces acidiscabies, Sporichthya polymorpha), Proteobacteria (Silicibacter sp. TrichCH4B,
Sorangium cellulosum, Rhodospirillaceae bacterium, Klebsiella pneumoniae), Bacteroidetes (Spirosoma fluviale, Phaeodactylibacter xiamenensi),
Archae (Halobiforma nitratireducens, Natronomonas pharaonis DSM 2160) and other Prokaryotes (Deinococcus radiodurans, Ktedonobacter
racemifer, Balneola vulgaris, Rubritalea squalenifaciens). Sequences include NOSs from various Eukaryotes such as Chlorophytes (Ostreococcus
tauri), Haptophytes (Chrysochromulina sp.), Stramenopiles (Thalassiosira oceanica, Thraustotheca clavata), Amoeba (Physarum polycephalum),
Heterobolosea (Naegleria gruberi), Fungi (Ophiocordyceps sinensis CO18, Rhizoctonia solani AG-1 IB, Verruconis gallopava, Colletotrichum orbiculare
MAFF 240422, Aspergillus flavus NRRL3357), Ichtyosporea (Sphaeroforma arctica). We used also NOSs from various animals such as Porifera
(Amphimedon queenslandica), Placozoa (Trichoplax B), Cnidaria (Stylophora pistillata, Nematostella vectensis, Hydra vulgaris), Bryozoa (Bugula
neritina), Brachyopoda (Lingula anatina), Annelid (Capitella teleta), Mollusc (Limulus Polyphemus, Sepia officinalis, Crassostrea gigas), Panarthropod
(Hypsibius dujardini), Arachnid (Ixodes scapularis), Collembolla (Folsomia candida), Crustacea (Marsupenaeus japonicas, Hyalella Azteca,
Daphnia pulex), Insect (Drosophila obscura, Pediculus humanus corporis, Zootermopsis nevadensis), Echinoderm (Acanthaster planci), Tunicate
(Ciona intestinalis), Cephalochordate (Branchiostoma floridae) and from various vertebrates such as Fish (Oncorhynchus mykiss, Callorhinchus
milii, Lepisosteus oculatus, Scleropages formosus, Latimeria chalumnae), Bird (Nestor notabilis), Crocodile (Alligator sinensis), Turtle (Chrysemys
picta bellii), Reptile (Python bivittatus), Amphibian (Xenopus laevis) and various Mammals (Dasypus novemcinctus, Monodelphis domestica,
Ornithorhynchus anatinus, Phascolarctos cinereus, Sarcophilus harrisii) including the three canonical NOSs from Mus musculus (iNOS), Bos taurus
(eNOS) and Rattus norvegicus (nNOS). The types of chosen isoforms (when several did exist) was made randomly to increase the heterogeneity of the
sampling. In several cases, multiple isoforms were used for a single species. Labels include the species names and the sequence length in order to ease
the identification of the NOS type. Phylogenetic branches were colored based on the type of NOSs (eNOS, nNOS and iNOS, but also Oxy-NOS, Glob-
NOS, EF-NOS and FeS-NOS) or on the nature of the clade (arthropods, fungi…). Sequence alignment of the full-length proteins has been achieved using
Jalview 2.7. © as multiple alignment editor (166) and PROBCONS © with two rounds of pre-training, 300 passes of iterative refinement and 3 passes of
consistency transformation (see supplementary file. Phylogenetic tree was generated using Seaview 4.5. © graphical interface using PhyML algorithm
(with Blosum62 model and 100 replicates bootstrapping).

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Revisiting the structure, function and evolution of NOS family

Figure 6. This phylogenetic tree does not aim at classifying the huge diversity of NOSs (that encompass several hundreds of proteins) but at illustrating
the difficulty in achieving this kind of analysis with a family of proteins that exhibit a strong diversity of molecular structures, catalytic functions in various
ecological and physiological conditions. In this NOS phylogenetic tree, NOSs are whether distributed along their specific structure or along the species
phylogeny. In vertebrates, NOSs are split in three branches that correspond to the three mammalian isoforms (eNOS, nNOS and iNOS). Surprisingly, this
distribution does not hold for the other chordates such as cephalochordates or echinoderms. Even if the invertebrates and the basal metazoan exhibit
various isoforms they do not partition between nNOS-like or iNOS-like but follow their species phylogeny. This suggests a completely distinct evolution of
NOS family outside the vertebrates’ lineage. Sometimes, NOSs gather along morphological/physiological similarities. This is the case for the NOS from
the oomycetes Thraustotheca clavata that cluster with the Fungi NOSs. Likewise, NOSs in the “Algae” branch belong to photosynthetic organisms from
completely distinct phyla: plants (Ostreococcus tauri), haptophytes (Chrysochromulina sp.) and stramenopiles (Thalassiosira oceanica). In prokaryotes,
NOSs first gather along their type of structures: Oxy, Glob-NOS, EF-NOS, Fe/S-NOS (see Figure 3), independently on the phylogenetic distribution of the
species. For example, the “Glob-NOS” branch includes NOSs from various classes of Cyanobacteria and Bacteroidetes, whereas the (EF-NOS-Fe/S-
NOS) branch correspond to Cyanobacteria and Proteobacteria. Apart for protists, this phylogenetic tree grossly overlaps with a species-based Tree of Life.

Thraustotheca clavata (a Stramenopiles species
too) is located within the branch of Fungi NOSs. The
same multiple rationales account for NOS phylogeny
in prokaryotes that balances between a structure-
based phylogeny (Glob-NOSs branch includes NOSs
from Cyanobacteria and Bacteroidetes, whereas
cyanobacterial EF-NOSs cluster with Fe/S-NOSs from
Proteobacteria; see below for structural explanations),
and species-based phylogeny (in the case of Archae or
Actinobacteria for instance). Besides, the distribution
of NOS is not homogeneous within most of these
clades, with a discrete presence in some phyla, and
major absences in other ones (see above). A rationale
that would try to address the evolution of the NOS
family as a whole would have to imply many additional
scenarii and many singular events (HGT, loss, gain…)
to account for such a heterogeneous distribution and
complex phylogeny.
in the NO° field and that imposes a unique mamma-
lian-centred vision of NOSs. Grossly, all new NOSs
are believed to behave like one of the three mam­
malian isoforms (see Figure 3). As seen through this
phylogenetic tree, this vision only holds for vertebrates
NOSs and has no heuristic value outside this phylum.
In fact, because of its very nature, an oxygenase
that uses a very sophisticated and sensitive redox
mechanism to produce a radical, gaseous, and thus
extremely reactive molecule (NO°) with a large array
of biological reactivity, NOS appears as a versatile
enzyme: no fixed biochemical activity can be assigned
to it, NOS biochemical activity can give rise to
various biological effects, depending on the cellular
or biological environment, these effects can lead to
distinct and often opposed biological outcomes. This
versatility has major implication on the way we should
investigate NOS structure, function and evolution.

We believe that NOS phylogeny and 4.2.1. NOS function

distribution cannot be explained by considering
all these NOSs as the same protein, and that this
phylogeny is unable reflect the evolutive history of this
family. We think that the only way to draw some sense
out of this picture is to consider several different groups
of NOSs, corresponding to strictly different (structural
and functional) types of proteins, and following distinct
evolutive tracks.
4.2. A singular versatile enzyme
The main difficulty when addressing NOS
evolution is linked to the “mammal bias” that prevails
There is some confusion in the way we
address NOS function. It often encompasses three
distinct phenomena: NOS chemical activity, the
biochemical effects of its catalytic production and their
ensuing biological function. NOS function has mostly
been understood as “NO°” function, whereas there
is no univocal relationship between NOS and NO°.
Indeed, NOSs have the ability to achieve different
chemistries, to produce various reactive species that
in turn exert distinct biological effects, which may be
employed for various purposes. The “problem” of plant
NO-Synthases illustrates this confusion very well (105,

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Revisiting the structure, function and evolution of NOS family
106). As NO° is a ubiquitous and important physiological
mediator in plants, the presence of a plant NOS was
beyond any doubt. This led to the publication of two
articles in Cell and Science, confirming the common
idea that plants were meant to have a NOSs. These
articles were soon retracted, and the sequencing of
numerous land plants genomes confirmed the absence
of a “genuine” mammalian-like NO-synthase (94). On
the opposite many algae and photosynthetic organisms
(stramenopiles, haptophytes, bacteria) harbor a NOS.
So why land plants would have lost NOS as they still
need and use NO°, what could be the function of plant
NOSs if not related to land plant NO° physiology?
This question seems paradoxical if one considers
a univocal relationship between NOS and NO°. But
there is no paradox if one considers that NO° can be
produced by many alternative sources and that NOS
might have other activities than producing NO°. One
must therefore clearly distinguish between NOS and
NO°; understanding NOS function requires to analyze
the nature of NOS catalytic production, the biochemical
effects of this RNOS, and their biological impact.
4.2.2. Instability of NOS activity and function
This phylogenetic tree is supposed to
account for the evolution of the structure of a
designated entity (NOS) in relation with its function in
a particular environment. It relies on a certain stability
of the “activity” that enacts the selection and evolution
process. However, NOS enzymatic activity is not stable
in time: NOS today, i.e. mammalian NOSs as we know
them, are oxydo-reductase that uses a gas (oxygen)
to produce another gas (nitric oxide), both major redox
reagents. As NOSs probably emerge long before the
Great Oxidation Event (GOE), in an anoxic and highly
reductive environment, it is natural to think that its
initial biochemistry was unrelated to the oxidation of
Arginine and that its activity was not NO° production
(107). This is reminiscent of the “exaptation” concept
proposed almost forty years ago by S.J. Gould and
R. Lewontin (108) and defined by Gould and Vrba
as “ such characters evolved for other usages – or
for no function at all – later coopted for their current
role” (109). Thus, the original NOS structure might
have served another (or no) purpose than the current
ones described in mammals. As a consequence, as
NOS environment has deeply evolved in the last three
billion years, NOS chemistry has probably changed
several times. Likewise, as NOS chemistry is strongly
related to its physico-chemical environment (O2
concentration, redox status, Nitrogen cycle…), NOS
activity is also a function of its biological milieu. As
NOS physico-chemical environment strongly varies
between, for instance, a halophilic Archae (such as
Halobiforma nitratireducens), an anaerobe Bacilli
(such as Anoxybacillus pushchinensis), an insect NOS
or a macrophage NOS, it is likely that their catalytic
activity will vary likewise (58, 61). In this regard, one
could wonder what could be the function of NOS in
151
anoxic organisms/tissues, or even if some of its
biological activity could still be related to specific
anoxic conditions. NOS catalytic activity is not fixed
as it varies with the physico-chemical conditions of its
milieu, that itself varies with the geochemical history
and with the ecological niche of the organism.
4.2.3. Overlaps of NOS activity
As NOS environment has experienced
many physico-chemical changes in the last three
billions of year, many different chemistries, effects
and functions have probably emerged in the course
evolution. This is illustrated by the various catalytic
activities of mammalian and bacterial NOSs: Arg
oxidation (signaling), NO° dioxygenase (110), RN0S
isomerisation (detoxification (66)), heavy production
of RNOS ((67, 111-113)), nitrite reduction (hypoxic
signaling, (64, 65, 114)) and etc. The balance is
determined both by the milieu and many sophisticated
molecular regulations and each NOS might be apt
to achieve different activities simultaneously (Figure
7). It is therefore extremely difficult to determine “ex
nihilo” which function each of these NOSs is actually
exerting. Besides, the evolution of conditions with time
also holds at the organismal level and the same “NOS”
(even as an individual protein) might experience
different catalytic activity. As a consequence of its long
history, each NOS is able to exert distinct biochemical
activities that could superimpose and lead to distinct
biological effects.
4.2.4. Multiplicity of NOS
This analysis becomes even more complex
when this evolution concerns not only one single
NOSs but several different types of NOS per
organism. Indeed, in mammals, three different NOSs
have been found, with different structures and various
– if not opposite – functions (115, 116). This standard
picture is found throughout the vertebrate clade but
does not hold beyond it. As we highlighted it, only one
NOS-related sequence is to be found in the genome
of most organisms such as insects, bacteria, fungi,
plants… However, many different patterns (of NOS
distribution) are found throughout the tree of life.
For example, mollusks harbor at least two types of
NOSs with different structures (NOSX1 and NOSX2
for example with 1387 and 1163 residues for Aplysia
californica). Whereas cnidaria species seem to
host only one NOS, Acropora digitifera harbor three
highly different NOSs (Table 3, Figure 8A). This is
the same for the placozoan Trichoplax adherens that
displays three similar NOSs. This heterogeneous
picture is not limited to metazoans but extends to
plants and bacteria. For example, two different NOSs
(one full-length and one truncated form) are found
in the genome of Ostreococcus tauri (Figure 8B).
Two extremely different NOSs are also found in the
cyanobacterial Crinalium epipsamum (Figure 8C),
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Revisiting the structure, function and evolution of NOS family
Figure 7. Illustration of the variability of NOS catalytic activity as a function of its environment and of the nature of its biochemistry.
whereas all the other cyanobacterial genomes harbor
only one NOS-related sequence. This co-existence of
different number and types of NOSs in many different
species suggests the co-evolution of parallel regimes
of activity and functions.
4.2.5. What does NOS stand for?
As we try to describe the function and
evolution of this family of proteins, we should wonder
which protein we are actually dealing with and address
the heterogeneity of this family that goes beyond the
mammalian NOSs. Indeed, NOS family consists of
many different proteins with sequence lengths between
230 and 1950 residues that are composed of various
modules and share only one conserved domain, the
oxygenase domain (see below for structural details).
As this domain only represents 15 to 20 % of the
functional holoenzyme, it might not be sufficient to
delineate a standard “NO-Synthase” protein. Besides,
the strong homology of these catalytic domain does
not imply that the chemistry is similar, and that the
activity remains identical. In any case, the NOS family
is not a structurally homogenous family. The disparities
in NOSs structure indicate likely variations in their
activity and function.
In this context, it seems difficult to analyse the
evolution of one “standard” NOS, when its number and
structure vary unpredictably between phyla and within
phyla, when its structure is not linked to a common and
single activity, producing NO°, and when this activity
152
remains variable in time and space. The numerous
and different molecular structures, the variety of their
chemistry (due to structural but also environmental
changes), the multiplicity of RNOS effects and thus of
NOS potential function impedes any straightforward
phylogenetic analysis.
4.3. The necessity of an original approach
The complex relationship between the
structure, the activity and the function of proteins within
the NOS family calls for an adapted phylogenetic
approach. As the genomic sequence of any NOS
does not correspond to a standard function, one must
classify and analyze more precisely this family of
proteins. This should be based on a better knowledge
of the structure that could help characterizing their
probable biochemical activity and might provide a
more suitable vision of their biological function. For
that matter one should take into account the great
diversity of NOSs structure and try to relate it to specific
patterns of activity. We’d like to present how this
approach could be achieved on three representative
phyla: Cyanobacteria, Algae and basal Metazoans.
5. DIVERSITY OF NOS STRUCTURES
5.1. A variable assembly of multiple modules
The diversity of NOS proteins resides not only
in the number of NOSs that are present in any organisms
but also in the types of NOSs that are identified. Until
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Revisiting the structure, function and evolution of NOS family

Figure 8. Examples of the evolutionary and structural diversity of NOS family for three phylogenetic groups. Phylogenetic trees have been generated
using the same procedure as in Figure 5; modules used to depict the structures of various NOSs are the same as in Figure 9. White rectangles represent
additional gaps observed in the protein sequence whereas colored rectangles correspond to large inserts. Panel A : Basal metazoan NOSs. Panel B.
Plant NOSs. Panel C. Cyanobacterial NOSs. Schemes and Figures

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Revisiting the structure, function and evolution of NOS family

Figure 9. Examples of different structural families of NOS proteins. Protein sequences of various NOSs are schematized as the arrangements of
various structural, catalytic or regulatory modules. All NOSs share a common oxygenase domain (blue). FMN domain is represented by a dark orange
Flavodoxin module (Flav.) whereas the FAD/NADPH domain is represented by a light orange FNR box. Calmodulin binding region is depicted by a light
grey rectangle (CaM), dark grey when the region is putative. Zn/S motif is associated to a light green box. Additional domains (see main text) comprise a
globin domain (light blue box) a PEF motif (EF-H, yellow) ) a Short-chain Dehydrogenases/Reductases motif (SDR, violet), a bacterioferritin-associated
ferredoxin domain ((Fe-S), pink), a kinesin domain (KIN, grey), a CAP-ED domain (CAP, violet), an ankyrin-repeat domain (Ank, green) and some
domains of unknown function (Unk, DUF…). Examples of NOSs (SsNOS, HsNOS…) are associated to each type of structure.

now, a short number of structural motifs involved in
the control of the structure and mechanism of NOSs
have been used to characterize the sequences of new
NOS-like proteins: the catalytic site that lies within the
oxygenase domain; the Calmodulin-binding domain; the
auto-inhibitory elements (AIEs) that regulate electron
transfer and control NO° production; the N-terminal
extension that determine subcellular localization (24).
Based on these patterns, four types of NOSs have
been used as “canonical” NOSs: i) neuronal NOS that
is a full-length NOS with an N-terminal PDZ domain
that allows nNOS to anchor partner proteins; ii) other
constitutive NOSs (such as eNOS) that mostly differ from
nNOS by their N-terminal extension (palmytoylation/
myrystoilation sites for eNOS); iii) inducible NOSs that
are deprived of N-terminal extension and lacks most of
the auto-inhibitory elements suggesting an unleashed
NO° production; iv) bacterial NOSs that consists only
of the truncated oxygenase domain (Figure 9). These
categories have been commonly used to achieve a first
and gross classification of NOS-like proteins. Based on
the first sequence analyses made on mollusks, insects
and other animals, many articles have considered
that metazoan NOSs would globally correspond to
mammalian NOSs and could be classified along these
categories (102) (101, 103). As a consequence, it was
proposed that metazoan NOS might have a common
ancestor, presumably a neuronal NOS (101). Though,
as noted by Andreakis and colleagues, “the pres-
ence of domains defining the three isoforms—PDZ
domain, inhibitory loop, myristoylation, and palmitoy-
lation motifs — was differently observed though not
always ascertainable” (101). It should also be noted
that the classification based on genomic data is insta-
ble as some of the available genomes remain partial
and the provided sequences are often truncated. Be-
sides, the potential existence of multiple transcription
initiation sites and of alternative splicing variants, as
suggested for Limax, Physarum, and Drosophila for
example (85, 86, 117, 118), calls for cautiousness
when analyzing the genomic data of these NOS-
like proteins. Indeed, although our census of NOS
seems to confirm the predominance of standard “full-
length” NOSs in metazoans (in tetrapods the three
mammalian isoforms are found ubiquitously, nNOS
and iNOS are found in fishes, and the cephalochordate
present several neuronal NOS-like proteins), a simple
classification based on mammalian NOSs categories,
does not provide a pertinent framework to apprehend
NOS family.
5.2. Existence of other types of NOSs
New types of NOSs, different from mammalian
NOSs are increasingly found. The first unconventional
NOS was found in a Gram- bacterium, Sorangium

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Revisiting the structure, function and evolution of NOS family
cellulosum (Sc56NOS, (89)). This Sc56NOS illustrates
three major discrepancies between the structure of
mammalian NOS and that of new NOS-related proteins.
Although these NOSs exhibit a strong homology of
their oxygenase domain with that of mNOS, they are
characterized by : i) a distinct electron transfer chain:
in that case the flavodoxin domain is substituted by
another electron purveyor, presumably a bacterioferritin-
associated ferredoxin domain (Bfd – (2Fe-2S), Figure
9); ii) the absence of major regulating domains such
as the CaM-binding domain the zinc tetrathiolate motif
and the N-ter BH4 (tetrahydrobiopterin) binding region;
iii) the presence of unexpected enzymatic or regulatory
units: a Short-chain Dehydrogenases/Reductases motif
(SDR) and a PEF motif (Penta-EF-hand family), that
suggest new and additional biochemical activity; iv) a
modified sequence topology, with a complete inversion
of the place of the whole electron transfer chain that
is here located on the opposite (N-Ter) side of the
oxygenase, suggesting a completely different fold and
remodeled electron transfer process. In this regard, it
becomes unreasonable to think that this enzyme could
behave as a genuine NO-Synthase.
This atypical picture can now be observed
for many other new NOSs. Indeed, the analysis of the
primary sequence of many prokaryotic NOSs reveal
the existence of a new structural family of NOSs that
are all devoid of the standard reductase domain and
present an Iron-Sulfur cluster as part of the electron
transfer chain. These NOSs, schematized in Fig.3
(Fe/S-NOS), seem evolutionary related (Figure 5).
Some present alternative reductase motifs but lack
the SDR motif, such as NOSs from the proteobacteria
Rhodospirillaceae bacterium TMED140 or Silicibacter
sp. TrichCH4B. Some NOSs do not harbor a PEF-
regulatory motif or, like the NOS from Hyphomonas sp.
TMED17, have alternative domains such as a kinesin-
associated domain or a CAP-ED domain (for Effector
Domain of the CAP family of transcription factors).
Many Fe/S-NOSs are found in cyanobacteria such
as Microcoleus sp PCC7113, Microcoleus vaginatus
FGP-2, Crinalium epipsamum (Oscillatoriales) or
Scytonema hofmanni UTEX B 1581 and various
Nostoc and Calothrix species (Nostocales). These
NOSs are highly homologous and are evolutionary
related (Figure 5). They exhibit the same Fe/S motifs
in the N-ter region, and the presence of an EF-Hand
motif that suggests some Ca2+ sensitivity (family EF-
NOS, Figure 9).
Symmetrically, another family of atypical
NOSs can be found in cyanobacteria. We and others
have identified another family of NOS that present the
same Oxygenase-Reductase combination, without
the CaM-binding or the zinc tetrathiolate motifs but
supplemented by a globin-domain and an undefined
N-ter extension (119). These NOSs are mostly found
in cyanobacteria with distinct morphological properties
(subsection I, III, IV) such as Nostoc sp PCC 7107
155
(NsNOS), Synechococcus sp PCC7335 (SpNOS)
and Scytonema hofmanni (ShNOS) (Figure 9). They
are also present in some Bacteroidetes species of the
Spirosoma genus.
But structurally different NOSs can even
be found in phyla more closely related to mammals.
This is the case for plant NOSs that are devoid of the
N-terminal Zn/S and BH4-binding region and do not
harbor a mNOS-like CaM-binding domain (119, 120).
We also report here the presence of a singular NOS
in the Stramenopiles Thraustotheca clavate with a
split reductase domain and an additional C-ter region
comprising a domain of unknown function (Figure
9). Truncated forms of standard NOSs are also
often observed, such as the ones found in the coral
Acropora digitifera, or in the algae Ostreococcus tauri.
Inversely, many NOSs present additional sequences
(Figure 8). For example, NOSs from Echinodermata
(Acanthaster planci or Strongylocentrotus purpuratus)
present large N-ter insertions (around 350 residues
between the oxygenase and the PDZ domains),
whereas NOSs from the algae Gonium pectorale
display a large (over 400 residues) C-ter extension
that includes an ANK motif (ankyrin repeats) and a
globin domain (Figure 8B).
This rapid overview of various structural types
of NOSs does not aim at producing an exhaustive list
of all potential types of NOSs to be found. We just
want to stress the great diversity of NOS structures
that is not limited to the three-canonical mammalian
NOSs. All these new regulatory or catalytic motifs,
along with their specific and different combinations, will
ineluctably modify the nature of the chemical activity of
these NOSs.
5.3. Types of NOSs are not uniform within
a simple phylogenetic group
Phylogenetic analyses of NOS most often
aimed at producing one common and global evolutive
story for this family of protein. This implies that these
NOSs belong to a single type of protein, at least in
the considered clade. If the NOSs present within the
tetrapod superclass are relatively homogeneous, this
is not the case in many other phyla where the number
and nature of NOSs is constantly heterogeneous. For
example, the nNOS-like NOSs are not the only NOSs
in the mollusks phylum: whereas bivalves exhibit only
nNOS-like proteins, cephalopods have another type
of constitutive NOS (without PDZ domain). Besides
gastropods harbor 4 different types of NOS: nNOS-
like proteins, two additional types of constitutive NOSs
devoid of PDZ domain plus one iNOS-like protein
(not shown). The same pattern can be observed in
cnidarians (Figure 8A). Whereas only a small part
of cnidarians harbors a NOSs in their genomes,
many different types of NOSs can be identified, such
as iNOS-like (Discosoma striata), an nNOS-like
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Revisiting the structure, function and evolution of NOS family
(Orbicella faveolata), both (Stylophora pistillata) or
various truncated forms (Acropora digitifera). In these
conditions, it seems extremely difficult to elaborate
an evolutive story common for all NOSs when there
are so many different types of NOSs, apparently
randomly distributed. For instance, as iNOS-like
proteins are found in basal metazoans such as
cnidaria and placozoa and are the sole NOSs found in
other eukaryotes such as plant, amoeba and fungi, it
seems unlikely that the common ancestor of metazoan
could be a neuronal NOS-like protein. In fact, the
heterogeneity of NOSs types and distribution within
the tree of life prevents any simple and straightforward
explanation of NOS family evolution.
The wealth of genomic data will ineluctably
increase the number of NOS types and the
heterogeneity of their distribution. The limited
mammalian categories already proved insufficient
to characterize and class these new NOSs in a
stable and heuristic manner that could provide with
a “simple” explanation for NOS evolution. There
is a need of a better classification of NOSs, based
on a more precise knowledge on the structure and
function of NOS. Until now, NOS classification has
been based on structural motifs related to subcellular
localization (PDZ, myristoylation sites) and electron
transfer regulation (CaM domain, autoinhibitory loop)
but none were related to NOS catalytic activity. This
absence of concerns about the fine structure of the
oxygenase domain is due to the conviction that any
oxygenase domain is structurally identical and will
uniformly product NO° for any new NO-Synthase
(Figure 6). In this regard, the knowledge acquired
on mNOS structure and function, and particularly on
their oxygenase domain, should be thoroughly used
to generate a more precise description of the various
distinct types of NOSs. It is not our goal here to describe
the subtle variations of the oxygenase domains of the
many hundreds of NO-Synthases. However, we will
describe this kind of analysis for three representative
examples: NOSs from basal metazoans, plants and
cyanobacteria
5.4. Strong disparities in the structure of
oxygenase domains
5.4.1. Basal metazoans
We have identified NOS-related sequences
in six cnidarians, in one porifera and in the placozoan
Trichoplax adhaerens (Table 3 and Figure 8A).
Different sequences were obtained for Trichoplax
(isoforms TaNOSA, TaNOSB and TaNOSC) and
Acropora digitifera (AdNOS1, AdNOS2, AdNOS3).
Although the retrieved sequences might be
artefactually truncated, their alignment shows that
they correspond to three different proteins (not
shown). It shows various N-ter-truncated regions
AdNOS2, AdNOS3 TaNOSA and TaNOSB, but a
156
significant extension for AdNOS1. We also observed
several gaps in the sequence of AdNOSs and
Exaiptasia pallida NOS (EpNOS). We focused on
the part of the oxygenase domain overlapping with
iNOS domain that we use as numbering reference.
Percentage of sequence identity of the oxygenase
domain of these NOSs with iNOSoxy range between
50% (AdNOS and HvNOS from Hydra vulgaris) and
60% (EpNOS, TaNOSB and NvNOS and OfNOS,
NOSs from Nematostella vectensis and Orbicella
faveolata TAB). AdNOS1 and AdNOS2, along with
TaNOSA and TaNOSC, seem closely related (PI
>80 %). A rapid phylogenetic analysis (Figure 8A,
see legend) confirms the homology information. It
stresses the peculiar behavior of TaNOSB (distant
from the other TaNOS isoforms but closely related
to iNOS) and of AdNOS3 (distant from the other
AdNOSs and the other Scleractinia NOSs and close
to NOSs from Corallimorpharia). It also highlights
the existence of four NOSs groups corresponding
to species phylogeny (Anthozoans, Hydrozoans,
Sponges and Placozoans) with no obvious link
with their structural specificities (PDZ domain,
truncations, N-ter extension…). We further refine
this gross analysis by looking at major residues of
the catalytic sites, involved in substrate binding,
heme environment, BH4 binding, dimer interface and
substrate channel (not shown). This comparative
analysis shows that all Arg-binding and BH4-binding
residues are conserved for these NOSs. Likewise,
the hydrophobic pocket that surrounds the heme
is conserved. No specific feature of the oxygenase
domain can discriminate between the NOSs of these
4 families. The same phylogenetic analysis was
obtained for full-length proteins, suggesting that
no specific oxygenase-related feature can account
for these distinct families and the particularity of
AdNOS3 and TaNOSB. Though, whereas all NOSs
exhibit the mNOS-like valine that allows NO release,
NOS from hydra vulgaris shows a substitution into an
Isoleucine, similarly to what has been reported for
bacNOS (121), which suggest that HvNOS is not fit to
release NO (61, 122).
5.4.2. Plants
NOSs are absent from land plants but were
identified in the genomes of 20 different algae, from
the Streptophyta and Chlorophyta phyla, distributed in
12 orders from 9 distinct classes (Table 2, (94, 95)).
Apart from the large N-Terminal extension of Gonium
pectorale NOS (see above), plant NOS exhibit a
homologous global structure (Figure 8B). We report
only a few number of gaps (100 residues in Volvox
aureus NOS sequence) but several insertions in
structurally and functionally relevant regions, such
as a 120-residues insert in Pteromonas sp. (PsNOS)
and a 53-residue insert for Chlamydomonas cribrum
(CcNOS). Focusing on the oxygenase domain, we
report a weak homology with murine inducible NOS
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Revisiting the structure, function and evolution of NOS family
(percentage of sequence identity (PI) between 32% for
CcNOS and 38% for Prasinophytes NOSs) with weak
intra-class (around 50% PI) or intra-phyla (below 50%
PI) homology. Despite the global sequence homology,
the phylogenetic analysis of these NOSs do not overlap
with a species phylogeny (Figure 8B). Although NOS
within major classes (Mammiellales, Chlamydomonales)
form distinct branches, some associations remain
unexpected such as the grouping of NOSs from
Streptophyta, Chlorophyceae and Trebouxiophyceae
species, or of NOSs from the Chlorophyceae and
Ulvophyceae species (Figure 8B). The analysis of
the oxygenase domain sequence shows the absence
of the Zn/S cluster and of the N-terminal BH4 binding
region (not shown, (95, 120)). Arg-binding residues
are well conserved except for Bolbocoleon piliferum
NOS (BpNOS) that shows large variations in the 340-
350 and 360-370 region (iNOS-derived numbering).
Likewise, the hydrophobic core that surrounds heme
binding is extremely well conserved for plant NOSs,
except again for BpNOS. Unlike any other NOSs, this
NOS also exhibits the bacterial NOS-like Isoleucine,
suggesting an incapacity to efficiently release NO (121,
122). Surprisingly NOS from Cosmarium subtidinum
(CsNOS) does not exhibit a Valine, nor a Leucine but a
Phenylalanine, which questions the actual functioning
of CsNOS. Apart from the highly conserved Arginine
(iNOS 375) that h-bonds with BH4 moiety, only few
residues involved in BH4 binding are conserved. This
is again the case for BpNOS and CsNOS that are
deprived of the essential W457 and F470 residues
(W457 is engaged in p-stacking interaction with BH4 ring
and controls its redox properties (123), and both W457
and F470 are engaged in H-bonds with BH4 moiety
that controls its precise positioning at the edge of the
heme (124)). NOSs from plants seem to form a distinct
family from mammalian NOS with no Zn/S cluster, no
CaM domain, a weak global homology and an altered
BH4-binding capacity. However, some of these NOSs
exhibit singular structural feature (CcNOS and PsNOS
display major insertions, BpNOS and CsNOS show
dramatic changes in core catalytic residues) that will
definitely affect their actual enzymatic functioning.
Besides, the phylogenetic tree of plant NOS displays
unexpected partitioning that does not coincide with
algae phylogeny. Alternative explanations are thus
required to address the evolution and functioning of this
peculiar NOS family.
5.4.3. Cyanobacteria
We have identified 35 different NOSs in various
cyanobacterial species and subspecies (Table 1). Many
different types of NOSs are observed, sometimes in
the same species. If most of the NOSs correspond to
the “oxygenase” isoform (Figure 9 and 5C), several
Glob-NOS and EF-NOSs have been also identified.
Additionally, truncated forms of these EF-NOS (without
the SDR domain) or of the Glob-NOS (without the
157
reductase or the globin domains) can also been noted.
A rapid phylogenetic analysis suggests three different
families: Glob-NOS, EF-NOS (and related) and Oxy-
NOSs (Figure 8C). Identity percentage of the shared
oxygenase domain with iNOSs range between 40
and 48 %, which suggests that cyanobacterial NOSs
are more closely related to mammalian NOSs than
plant NOSs. All Glob-NOSs (and EF-NOS alike) are
extremely similar, despite the fact that the species
belong to very different branches of the cyanobacteria
tree and to distinct morphological subsections. This
is confirmed by the analysis of crucial catalytic site
residues (not shown). Whereas most of the Arg-binding
residues are conserved, the mammalian NOS-like
Valine is only observed in EF-NOSs whereas all the
Glob-NOS and most of the Oxy-NOSs instead exhibit
an Isoleucine that prevents NO° release. Likewise,
Glob-NOSs exhibit a slightly less well-conserved heme
hydrophobic pocket but a more conserved BH4-binding
capacity. The C-ter region of all these NOSs is clearly
remodeled with a more open heme edge (alike what
was observed for plant NOS). Along with the truncation
of the N-ter region, this surely affects BH4 binding.
This comparative analysis of major structural motifs
of the oxygenase domains of cyanobacterial NOSs is
in line with the phylogenetic analysis, which suggests
a diverging evolution of the three major families of
cyanobacterial NOSs within the phyla. The distribution
of each type of NOSs in all kinds of phylogenetic/
morphological groups of cyanobacteria could imply that
these three very different NOSs were already present
in the cyanobacterial common ancestor.
It is obviously impossible to achieve an
exhaustive analysis of every structural variations that
occur for all NOSs. However, using three different
model clades (from cyanobacteria, plants, and
cnidaria) we highlighted the facts that oxygenase
sequences were experiencing many different sorts of
truncations, gaps or inserts, but also many important
single-residue modifications that surely would affect
substrate and cofactor binding, and in turn NO°
synthesis and release. This kind of analysis is just an
example that should be extended to the many other
residues/motifs that are crucial in the chemistry of NO
synthesis.
6. DISCUSSION: DIVERSITY OF FUNCTIONS
This article aimed at showing the complexity
that lies behind NO-Synthases. We wanted to stress
the importance of the relationship between the
molecular structure of any single NOS, its function, its
environment and the evolution of the whole NOS family.
6.1. A Name is not a function
Genomics has modified the way we address
the question of function and evolution of proteins. It
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Revisiting the structure, function and evolution of NOS family
Figure 10. Schematic representation of NOS catalytic site and of the structural parameters that control NOS mechanism. 3D structures derived from
iNOS 1NOD PDB structure.
has allowed the identification of hundreds of proteins
that were automatically annotated “NO-Synthases”
based on the presence in their sequence of the
oxygenase domain. This oxygenase domain, that is
specific to NOSs, is highly homologous between all
these proteins, from bacteria to humans, which led to
the assembly of this family of proteins. Based on the
great knowledge gathered over the last thirty years
on NO° and NO-Synthases in mammals, all these
proteins are today genuinely (but improperly) believed
to catalyze the production of NO and to participate to
signaling functions.
The problem resides here in the belief that
a strong (but uncomplete) sequence homology – and
most of the time a simple database annotation – can
characterize the nature of such proteins and lead to
the assumption that all these proteins carry similar
structure and functions. Though, a name does not
make a function, not even a structure. The name “NO-
Synthase » hides a great diversity of objects. The
NOS family gather very different kinds of proteins that,
behind a common catalytic site, are characterized by
a great structural heterogeneity. Variations include the
absence of crucial regulatory modules, overhaul of
the electron transfer chain – including new reductase
domains, modified 3D architecture… – new regulatory
motifs and even new catalytic domains. In this regard,
it is reasonable to suggest that these NOSs are not
genuine NO-Synthases and might carry a completely
different activity and function.
158
6.2. A Structure is not a function
The high structural homology of NOS
catalytic site is believed to preserve a common
enzymatic activity between all these NOSs, namely
producing NO°. This is where the special nature of NO-
Synthases comes into line. Catalysis of NO synthesis
is not just a question of structure that would naturally
produce NO from its substrates L-arginine, NADPH
and dioxygen. This redox chemistry requires an
extraordinarily sophisticated mechanism that involves
a fine regulation of the proximal and distal heme
environment along with a complex sequence of proton
and electron transfers (Figure 10, (30, 125, 126)).
6.2.1. A built-in versatile catalysis
At first, it is to be noted that NOSs catalyze
two sequential and different sets of reactions (Figure
2). The hydroxylation of L-arginine involved a P450-
like mechanism (38, 127), whereas NOHA oxidation
is achieved through a NOS-specific mechanism (26,
32). The molecular mechanisms of these two reaction
sequences are clearly different and require different
processes of electron and proton transfers. The
necessary balance between these two reaction steps
is allowed by the rigid structure of the catalytic site that
ensures an adequate substrate positioning (distinct
between Arg and NOHA) within a complex distal
H-bond network, which in turn controls the switch
between a P450- (Step 1) and a NOS-specific (Step
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Revisiting the structure, function and evolution of NOS family
2) mechanism (30, 42, 63, 128-130). Subtle structural
changes might modify this the redox chemistry and
divert NOS from NO° production (131). Besides, NO
geminate recombination on NOS heme leads to the
co-existence of two distinct catalytic cycles (56, 58): an
NO°-producing cycle, that corresponds to an efficient
NO° release, and an RNOS-releasing cycle linked to
the reduction of heme-bound NO and its conversion
into other RNOS (such as peroxynitrite (PN) or nitrate).
We and others have shown that small variations in
some central catalytic parameters can modify the
balance between futile and efficient cycles, and
deeply modify NOS biochemical activity. Here-again,
small structural variations might modify the extent of
geminate recombination (132), the rates of ET (133,
134) or NO° release (121, 122) and lead to different
catalytic production (61, 110).
6.2.2. A highly-sensitive chemical system
The impact of negligible structural changes
on NOS activity can be illustrated by two other minor
modifications. We showed that subtle changes in
H-bonding that can be observed between bacNOS
and mNOS, modify the balance between an effective
O2 activation and a futile autoxidation (135, 136).
Small variations in the H-bond between the proximal
thiolate and the vicinal tryptophan could slow down
NOS activity or turn it into a superoxide anion synthase
(137). An even more subtle variation in the structure can
radically modify NOS activity. A ValàIle substitution,
that would go unnoticed in phylogenetic analyses,
switch one protein from a NO° synthesis device, to
a RNOS (nitrate but perhaps peroxynitrite) synthase.
Indeed, this mutation slows down NO° off rate by two
orders of magnitude, preventing NO° efficient release
and favoring its dioxygenation (66, 121). It has been
shown that this simple mutation in iNOS prevents
NO° synthesis and switch it to nitrate synthesis (61,
110). All the NOSs that carry this V/I substitution thus
appear unfit to produce NO°, not mentioning the ones
that exhibit another type of residues (Ala, Phe). The
proton transfers have been poorly investigated and
the exact number, sequence and source of protons
remains undetermined. It is known however that an
impaired proton transfer during Step1 would prevent
the build-up of an Oxoferryl complex (necessary for
Arg hydroxylation) and lead to the production of H2O2.
Thus, small changes in the distal H-bond network,
or in the substrate binding modes could convert NO-
Synthases into H2O2 producing enzyme (138).
6.2.3. Electron transfer (ET) as a major NOS
fingerprint
The electron transfer processes have been
thoroughly investigated and are today much more
documented (34, 36, 44). NOSs are characterized by
two complementary electron donors: i) the reductase
159
domain is the original source of the electrons required
to reduce the heme and the BH4 radical. Despite strong
homology, the reductase domains of mammalian
NOSs harbor different properties, leading to major
differences in the electron transfer rates (133). We
and others showed that NO° production is the result
of a fine tuning between ET and other catalytic
parameters and that changes in electron transfer rate
modify NOS catalysis outcome (58, 63). Obviously,
the new reductase motifs that we described here-
above, or even external reductase proteins, will show
a completely different tuning of ET rate (if any), leading
to alternative catalysis.
Another major marker of NOS mechanism
is the use of a redox cofactor, BH4. Indeed, NO°
synthesis by NOS requires a fast ET (to prevent FeIIO2
autoxidation, (36)), but a transient and reversible one,
in order to preserve the capacity to release NO° (a
fast ET would lead to NO-/PN/NO3- release (36, 130)).
This peculiar ET is provided by BH4 that activates
FeIIO2 intermediate but re-oxidizes Fe-NO complex
to allow NO° release. In this regard NOSs that do not
harbor the same BH4 association should be unable
to produce NO°. Though, many bacteria that harbor
a NOS lack the BH4 biosynthesis machinery. It has
been proposed that BH4 could be replaced by another
redox cofactor, such as tetrahydrofolate ((139), FH4).
Though, data on FeIIO2 activation by FH4 are lacking,
such as the actual role of FH4 in NO° production.
On the opposite, we observe here a strong variation
of BH4-binding elements. Apart from the Arg375, the
N-ter binding region and the C-ter elements involved
in BH4 binding site are generally not conserved in non-
metazoan NOSs, suggesting that BH4 or even other
redox cofactor might not intervene in NOS catalysis.
As shown here, NOS molecular mechanism
is extremely complex and versatile. Although all NOS
proteins exhibit similar structure for the oxygenase
domain, they do not automatically carry on the same
chemistry, and their function might be extremely
variable.
6.3. An Activity is not a function
6.3.1. The role of environment in NOS/NO activity
NO-Synthases are very peculiar oxygenase
proteins. Beyond being a redox protein, NOSs have
the particularity to catalyze the conversion of one gas
(O2) into another gas (NO°) through an extremely
sophisticated redox mechanism, which confers to
NOS a strong dependency to its physico-chemical
environment. For instance, NOS activity will greatly
depend on the redox status of its close cellular
environment: oxidative conditions (inflammation,
immune responses…) will lead to the oxidation of NOS
pterin cofactor that will turn NOS into a superoxide
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Revisiting the structure, function and evolution of NOS family
synthase (140-142). NOS activity also depends on O2
cellular concentrations: we showed that NOS apparent
KmO2 was greatly different between mammalian NOSs
(from 2 to 400 µM), suggesting different sensitivities to
O2 concentration changes and different biochemistry
in hypoxic, and even anoxic conditions (55). As an
illustration, it has been proposed that eNOS, in hypoxic
conditions, will maintain its role in vasodilation by
producing NO° by other means: in these conditions,
NOS could convert significant concentrations of nitrite
into NO° by achieving another kind of chemistry, Nitrite
reduction (114). Symmetrically, in oxidative stress
conditions, NOS activity could be switched to other
enzymatic activity such as catalase in the presence of
high concentrations of H2O2 (72), PN isomerase (66) or
NO° dioxygenase when high NO° concentrations lead
to direct major NO° binding to NOS heme (60, 61, 110).
Beyond NOS multiple faces, the adaptive
evolution of NO° function has been already described
(143). As NO° is a diffusible and extremely reactive
gas, its biochemical activity will mostly depend on its
biological environment: i) NO first emerged in a re� ­ NOS is believed to produce NO°, “NO°” name is
ductive environment and was most likely mobilized in
antioxidant defense (107, 143). This is still the case
today in mammals, where NO° is a major contributor
to the redox homeostasis, prevents oxidative stress in
the cardiovascular system (115); ii) in relation with its
anti-oxidant use and its gas properties, NO° eventually
became the central element of a sensing/signaling
machinery; at distinct organismal/cellular level, NO°
plays the role of a signaling molecule mobilized in
intercellular signaling or symbiosis regulation (98,
99, 144-148); iii) in a more oxidative context, NO
potentiates the reactivity and toxicity of many RNOS
(such as superoxide anion), leading to alternative uses
in immunity. It clearly appears that NO° is involved in
a large range of physiological processes that extends
beyond its native function in mammals. However, this
variation of NO° biochemistry does not always take
place in a selected/evolutive frame but is often at the
core of various pathological dysfunctions. In mammals
for example, oxidative conditions will lead to the un-
coupling of NOSs, switching its biological activity from
signalling to inflammation (149). It is therefore reason�
- WHICH EVOLUTION FOR NOS?
able to assume that the variability of the ecological and
physiological environment of NO° might have led to a
broadening of its biological function.
This statement must extend to the large pan-
el of reactive nitrogen and oxygen species (RNOS),
deriving from NO° bio-synthesis, such as peroxynitrite
(150-152) or nitrosothiols (153, 154). Indeed, each
RNOS displays a specific and distinct chemical biol-
ogy (155, 156), that was first evoked to explain the
multiplicity of “NO°” biological role (150). However the
« good » RNOS, such as NO° and nitrosothiols, have
been increasingly associated to pathological activity
(157-159), whereas the “ugly” ones such as nitrogen
160
dioxide or peroxynitrite are tentatively associated to
signalling processes (160). Biological effects of RNOS
are sequentially depending on their reaction with oth-
er biomolecules (and conversion into different RNOS),
on their interaction with their physiological targets, on
the cellular and metabolic status of the organisms, and
more globally on the physico-chemical properties of
their milieu.
To summarize, NOS is able to catalyze the
production of multiple RNOS. The balance between
these distinct catalytic reactions is tuned by the
properties of the milieu. At a second level, outcome
of NOS activity is made more complex by the various
and variable reactivity of each of these RNOSs. At
last, the pattern of RNOS production and reaction
is itself controlled by the physiological status and
the physico-chemical environment of NOS. This
incredibly complex biology of NOS is often screened
by the confusion that persists in the apprehension of
NOS, NO° and RNOS. In most of the reports, there is
a confusion in the respective nature of these agents:
actually recovering many different nitrogen oxides.
This is illustrated by the extensive and maladroit use
of DAF as a NO° probe, and of cPTIO as an NO°
inhibitor that overlooks the actual current chemistry of
cPTIO with NO° and of DAF with other RNOS. If we
assume that “NOS = NO° = RNOS”, there is no way
to address the diversity of NOS and NO° biological
effects and their interaction with its physiological
and chemical environment. NOS function is not
all included in its genomic sequence nor in its 3D
structure but in the complex interaction between its
mechanism, the RNOS chemistry and the physico-
chemical environment. If we want to address NOS
function and its evolution, we must take into account
this complex diversity of NOS structure, activity and
effect, and systematically investigate the relationship
between NOS structure, its enzymatic activity, the
biological effect of its catalytic production and the
impact of its environment at each level.
7. CONCLUSION: WHICH FUNCTION AND
After this series of questionings, it appears
difficult to assign a definitive function to any new NOS.
Is there a unique, selected function or a multiplicity of
biochemical activity? When mammalian NOSs are to
be considered, one would unambiguously respond:
one function, signaling versus immunity. However,
iNOS might be involved in the signaling and promotion
of the immune response. eNOS and nNOS might be
both exerting local antioxidant activity. In pathological
conditions, constitutive NOSs will eventually be
involved in deleterious biochemical processes. The
frontiers between distinct functions, and beyond that
between pathological and physiological effects, do
© 1996-2019
Revisiting the structure, function and evolution of NOS family
not seem so obvious when it comes to NOS and NO°,
even in mammals. This could have been foreseen as
NO° emerged as an atypical signaling molecule, a new
type of mediator: “a gas was indeed a surprise for an
endogenous role, and a labile and toxic gas even more
so. As the first surprise of such an unlikely agent” as
it was highlighted by D.E. Koshland in its editorial of
the 1992 Science Molecule of the Year (9). Unlike the
conventional signaling mediators, NO° was a toxic,
labile and extremely reactive molecule that thirty years
after its discovery seems to exert its signaling activity
in an uncontrolled and even random manner. Its role
in signaling processes along with its strong and toxic
reactivity could have led to reconsider the definition of
“Signaling” and even to question the frontiers between
a physiological activity and a noxious role, between
transducing a signal and exerting deleterious and
irreversible modifications. If we consider the large
biological chemistry of RNOS and the versatility of
NOS enzymology, these questions become even
more puzzling. The multiple facets of NOS structure
and activity could be addressed by considering its
structure, and in particular the oxygenase domain, as a
template that can catalyze various enzymatic reactions
and that can be used for different purposes. In this
context, the function seems undeniably related to
both the enzymatic possibilities of NOS, to its physico-
chemical environment and to the biological conditions.
These three levels should be tackled simultaneously in
order to assign a function in the frame of a biological
purpose.
Although environment adaptation might have
driven the evolution of NOS structure, the versatility
of its enzymology, the complexity of RNOS biological
chemistry and overall its impact on its own cellular/
biological environment might make the rationale(s)
of such an evolution difficult to unveil. The loose
connection between Structure and Function makes
the phylogenetic approach much more arduous. The
superimposition of many functional stories, that cross,
overlap and co-act impedes a simple straightforward
analysis of NOS phylogeny. Which specific NOS
activity is to be focused on when a new activity (and
function) emerges and co-exists with an already
efficient one? What is the selected structure and for
which evolutive gain? Considering the important
diversity of NOS structures and the multiplicity of their
potential activity, NOS phylogenetic analysis will have
to disentangle many parallel evolutive stories and
address the co-evolution of distinct structure/activity
patterns. We believe that this complex structure-
function-evolution relationship is not unique to NOSs.
What we describe here for NOSs should apply to many
other proteins, and in particular to metalloproteins that
are more susceptible to carry multiple biochemical
capacities. This is illustrated for example by Globins
that display a large variety of biochemical activities (O2
storage and transport, signaling, NO detoxification…)
161
that are related both to fine tuning of their active sites
and to their physiological and ecological environment.
In this context, a specific methodological
approach seems needed to tackle the complex
relationship between NOS structure, catalytic
functioning, environmental niche and biological
function. The large diversity of NOS family precludes
any systematic exploration and necessitates the use
of model families that display significant structural
diversity. By combining modelling studies, structural
characterization, and in vitro/in vivo functional
investigations, an integrative and interdisciplinary
approach might give some insight into the structural
and ecological parameters that determine NOS
activity, and provide with some predictions about the
function of any different NOS protein. This task will
also have to take into account the ever-changing
environment of NOS, through time but also milieu. The
role of NOS milieu here is major for it not only serves
as a selection riddle, but for it conditions NOS actual
activity. NOS environment will determine its activity, its
function and the way NOS structure will evolve. This is
complementary to the concept of exaptation as here
the environment might contribute to the fashioning of
“aptation”, participate to the design of the function and
co-determine its evolution.
In any case, we believe it is now timely to ask
what NOS stands for. In order to answer this question,
we must avoid reducing the categories by which we
apprehend NOS to the sole mammalian concepts
and try to write complex evolutive stories that are
not restricted to a small number of NOS archetypes
but that take into account the vast multiplicity of NOS
structure and function, and their singular interaction
with their environment.
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Abbreviations: Nitric Oxide (NO°); NO-Synthases

(NOSs); Nitrous oxide, N2O; Endothelium-Derived
Relaxing Factor (EDRF); neuronal NOS (nNOS);
endothelial NOS (eNOS); inducible NOS (iNOS);
Calmodulin-binding region (CaM); Electron
transfer (ET); Auto-Inhibitory Elements (AIE);
peroxynitrite (PN); bacterial NOSs (bacNOS);
mammalian NOS (mNOS); horizontal genome
transfer (HGT); Great Oxidation Event (GOE);
Reactive Nitrogen and oxygen Species (RNOS);
Reactive Oxygen Species (ROS); L-arginine
(Arg); Tetrahydrobiopterin (BH4); tetrahydrofolate
(FH4); All NOSs from various species are labelled
XyNOS where X and y are the initial of the species.

Key Words: NO-Synthases, Nitric Oxide,

Phylogeny, Mechanism, Function, Evolution,
Review

Send correspondence to: Jerome Santolini,

Laboratoire Stress Oxydant et Détoxication,
Institute for Integrative Biology of the Cell (I2BC),
CEA, CNRS, Univ Paris-Sud, Universite Paris-
Saclay, F-91198, Gif-sur-Yvette cedex, France,
Tel: 33-1-69-08-53-63, Fax: 33-1-69-08-87-17,
E-mail: jerome.santolini@cea.fr

171 © 1996-2019